Solid forms for tissue repair

ABSTRACT

This invention provides aragonite- and calcite-based scaffolds for the repair, regeneration, enhancement of formation or a combination thereof of cartilage and/or bone, which scaffolds comprise at least two phases, wherein each phase differs in terms of its chemical content, or structure, kits comprising the same, processes for producing solid aragonite or calcite scaffolds and methods of use thereof.

BACKGROUND OF THE INVENTION

Surgical intervention and grafting are sometimes necessary to restoremechanical function and reconstruct the morphology of bone andcartilage, resulting from trauma, tumors, or abnormal bone developments.

Synthetic materials such as metals and bone cements have also been usedfor restoring and reconstructing bone for many years, but often resultin stress-shielding to the surrounding bone and fatigue failure of theimplant. Another possibility is autologous bone grafting, although thesupply of autologous bone tissue is limited and its collection ispainful, with the risk of infection, hemorrhage, cosmetic disability,nerve damage, and loss of bone function. In addition, significantmorbidity is associated with autograft harvest sites. These problems maybe overcome by engineering tissue using scaffolds made of synthetic ornatural biomaterials that promote the adhesion, migration,proliferation, and differentiation of bone marrow stem cells, also knownas mesenchymal stem cells (MSCs). An association between biocomponentsand biologic regenerative and repair responses can be promoted byproviding a scaffold containing spaces morphologically compatible withosteons and their vascular interconnections.

The immediate microenvironment and the three-dimensional (3D)organization are important factors in differentiation in general andparticularly in chondrogenic and osteogenic differentiation.

Some bone tissue engineering scaffolds consists of natural polymers,such as collagen, alginate, hyaluronic acid, and chitosan. Naturalmaterials offer the advantages of specific cell interaction, easyseeding of cells because of their hydrophilic interactions, low toxicityand low chronic inflammatory response. However, these scaffolds oftenare mechanically unstable and do not readily contribute to the creationof tissue structures with a specific predefined shape fortransplantation. To obtain mechanical strength, chemical modification isrequired, which may lead to toxicity.

Defects and degeneration of the articular cartilage surfaces of jointscauses pain and stiffness. Damage to cartilage which protects joints canresult from either physical injury as a result of trauma, sports orrepetitive stresses (e.g., osteochondral fracture, secondary damage dueto cruciate ligament injury) or from disease (e.g. osteoarthritis,rheumatoid arthritis, aseptic necrosis, osteochondritis dissecans).

Osteoarthritis (OA) results from general wear and tear of joints, mostnotably hip and knee joints. Osteoarthritis is common in the elderlybut, in fact, by age 40 most individuals have some osteoarthitic changesin their weight bearing joints. Another emerging trend increasing theprevalence of osteoarthritis is the rise in obesity. The CDC estimatesthat 30% of American adults (or 60 million people) are obese. Obeseadults are 4 times more likely to develop knee OA than normal weightadults Rheumatoid arthritis is an inflammatory condition which resultsin the destruction of cartilage. It is thought to be, at least in part,an autoimmune disease with sufferers having a genetic predisposition tothe disease.

Orthopedic prevention and repair of damaged joints is a significantburden on the medical profession both in terms of expense and time spenttreating patients. In part, this is because cartilage does not possesthe capacity for self-repair. Attempts to re-grow hyaline cartilage forrepair of cartilage defects remain unsuccessful. Orthopedic surgery isavailable in order to repair defects and prevent articular damage in aneffort to forestall serious degenerative changes in a joint. The use ofsurgical techniques often requires the removal and donation of healthytissue to replace the damaged or diseased tissue. Techniques utilizingdonated tissue from autografts, allografts, or xenografts are whollyunsatisfactory as autografts add additional trauma to a subject andallografts and xenografts are limited by immunological reactivity to thehost subject and possible transfer of infective agents. Surgicalattempts to utilize materials other than human or animal tissue forcartilage regeneration have been unsuccessful.

An ideal material which restores mechanical function and reconstructsthe morphology of bone and cartilage is as yet, lacking.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides coralline-basedscaffolds for inducing or enhancing repair, regeneration or enhancementof formation of cartilage or bone, or a combination thereof, whereinsaid scaffold comprises aragonite or calcite.

In some embodiments, the invention provides a scaffold for tissuerepair, said scaffold consisting essentially of two phases wherein:

-   -   a first phase of said two phases comprises solid coral or        biolattice comprising hyaluronic acid; and    -   a second phase of said two phases comprises solid coral or        biolattice and said second phase further comprises a series of        hollows along a longitudinal axis in said second phase.

In one embodiment, the present invention provides a scaffold for repair,regeneration or enhancement of formation of cartilage or bone, or acombination thereof, which scaffold consists of a solid form ofaragonite or calcite and further comprises:

-   -   at least a first phase, comprising voids having an average        diameter ranging from about 60-160 μm; and    -   at least a second phase, comprising voids having an average        diameter ranging from about 170-850 μm.

In some embodiments, according to this aspect, the scaffold furthercomprises a third phase, comprising voids having an average diameterranging from about 170-300 μm and said second phase comprises voidshaving an average diameter ranging from about 350-850 μm and said thirdphase is positioned between said first and second phase.

In another embodiment, the invention provides a scaffold for repair,regeneration or enhancement of formation of cartilage or bone, or acombination thereof, which scaffold consists of a solid form ofaragonite or calcite and further comprises:

-   -   at least a first phase, comprising pores having a pore volume        (porosity) ranging from about 35-55%; and    -   at least a second phase, comprising pores having a pore volume        (porosity) ranging from about 56-95%.

In some embodiments, according to this aspect, the scaffold furthercomprises a third phase, comprising pores having a pore volume rangingfrom about 56-80%, wherein said second phase comprises voids having porevolume (porosity) ranging from about 81-95% and said third phase ispositioned between said first and second phase.

In another embodiment, this invention provides a scaffold for therepair, regeneration or enhancement of formation of cartilage, bone, ora combination thereof, which scaffold consists of a solid form ofaragonite or calcite isolated from a coral and further comprises:

-   -   at least a first phase, comprising voids having an average        diameter, pore volume or combination thereof, which corresponds        to that of the native coral from which said solid form was        isolated; and    -   at least a second phase, comprising voids having an average        diameter, pore volume or combination thereof, which average void        diameter, pore volume or combination thereof is greater than        that of said first phase by from about 15-100%.

In some embodiments, according to this aspect, the scaffold furthercomprises a third phase, comprising voids having an average diameter,pore volume or combination thereof, which average pore diameter, porevolume or combination thereof is greater than that of said first phaseby from about 15-35% and said second phase comprises voids having anaverage diameter, pore volume or combination thereof, which average porediameter, pore volume or combination thereof is greater than that ofsaid first phase by from about 40-100% and said third phase ispositioned between said first and second phase.

In another embodiment, this invention provides a process for thepreparation of a multi-phasic scaffold for inducing or enhancingcartilage or bone formation or repair, or a combination thereof, saidprocess comprising the steps of:

-   -   contacting only a portion of a solid form of aragonite or        calcite with a calcium chelator and an acid to yield a solid        form comprising enlarged voids in at least a portion of said        solid form; and    -   washing and drying said solid form under applied negative        pressure.

According to this aspect, and in some embodiments, the contacting isconducted over a duration and under conditions, which vary as aconsequence of the desired final geometry of the scaffold.

According to this aspect, and in other embodiments, the solid formproduced by said process comprises at least two phases, which phasesdiffer in their pore volume (porosity), or which phases comprise voidswhich differ in terms of the average diameter of said voids, or acombination thereof.

In some embodiments, this invention provides a scaffold producedaccording to a process of this invention.

In one embodiment, this invention provides a scaffold for repair ofcartilage comprising a biolattice consisting essentially of calcitecapable of being inserted within a site of cartilage repair. In someembodiments, the biolattice is derived from Tetraclita rufotincta.

In another embodiment, this invention provides a scaffold for tissuerepair, said scaffold comprising at least two phases wherein a firstphase of said two phases comprises a coral or biolattice and a secondphase comprises a biocompatible polymer.

In one embodiment, this invention provides a method of inducing orenhancing cartilage or bone formation or repair, or a combinationthereof, said method comprising implanting in a subject, a scaffold ofthis invention within a site in need of cartilage or bone formation,repair or a combination thereof.

According to this aspect, and in some embodiments, the method comprisesexposing said site in need of cartilage or bone formation or repair or acombination thereof, and optionally exposing bone tissue locatedproximally to the site of cartilage repair in said subject in need ofcartilage repair or regeneration, prior to implanting said scaffold.

According to this aspect, and in some embodiments, the method forinducing or enhancing cartilage repair or regeneration comprises thestep of affixing at least a portion of said scaffold within bone locatedproximally to said site of cartilage repair

In another embodiment, this invention provides a process for thepurification of coralline-based scaffolding, said process comprising thesteps of:

-   -   contacting solid aragonite of a desired size and shape with a        solution comprising an oxidizing agent; and    -   washing and drying said solid aragonite        whereby one or each of said steps is conducted under applied        negative pressure.

According to this aspect, and in some embodiments, the process comprisesconducting said contacting under mildly acidic conditions. In someembodiments, the solution comprises sodium hypochlorite.

According to this aspect, and in some embodiments, the process furthercomprises subjecting the solid aragonite to a temperature of at least275° C. under applied negative pressure.

In some embodiments, the applied negative pressure ranges between about0.2 to 0.00001 Bar, or in some embodiments, the applied negativepressure ranges between 0.4 to 0.0000001 Bar.

In some embodiments, the invention provides a coralline-basedscaffolding produced by the process according to this aspect of theinvention.

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference. In case of a conflict between thespecification and an incorporated reference, the specification shallcontrol. Where number ranges are given in this document, endpoints areincluded within the range. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or sub-range withinthe stated ranges, optionally including or excluding either or bothendpoints, in different embodiments of the invention, to the tenth ofthe unit of the lower limit of the range, unless the context clearlydictates otherwise. Where a percentage is recited in reference to avalue that intrinsically has units that are whole numbers, any resultingfraction may be rounded to the nearest whole number.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts positioning an implant within a site forcartilage and bone repair, where the scaffold 1-10 is positioned suchthat a first phase is positioned within the cartilage and a second phaseis positioned within proximally located bone.

FIG. 2 depicts successful incorporation of embodiments of a scaffold ofthis invention within a cartilage and bone defect. FIG. 2A shows aphotograph of a drilled hole in a medial femoral condyle of a goat; D=6mm, L=7.5 mm. FIGS. 2B and 2C show a photograph of implantation of thescaffold within the site of injury. FIG. 2D shows that at 19 days postimplantation, the implant was successfully incorporated within thecartilage, signs of vascularization and an intact meniscus are seen.

FIG. 3 shows light micrographs of osteochondral tissue in which anembodied scaffold of this invention has been implanted. FIG. 3A shows alow magnification (2×) 9 weeks after implantation of an embodiedscaffold described herein in a medial femoral condyle of a goat; D=5.2mm, L=7.5 mm; visualized with standard H & E staining. FIG. 3B shows acomparable section of the tissue stained with Masson Trichrome. Notethat the area of implant, (highlighted by the dotted rectangle in 3A) isreplaced by woven bone and cartilage.

FIG. 4, similar to FIG. 3 shows light micrographs of osteochondraltissue in which an embodied scaffold of this invention has beenimplanted, subjected to different staining protocols. FIG. 4A presents alow magnification (2×) of the tissue 9 weeks after implantation in amedial femoral condyle of a goat; D=5.2 mm, L=7.5 mm D=5 stained withSafranin O, and FIG. 4B shows a comparable section stained for detectionof Collagen type II. FIG. 4A demonstrates the presence of a homogeneousred band of cartilage covering normal bone and the area of defect.Similarly, FIG. 4B shows positive staining for collagen type II alongthe band of cartilage covering the defect (and adjacent normal cartilageas an internal positive control).

FIG. 5 shows light micrographs of osteochondral tissue in which anembodied scaffold of this invention has been implanted, subjected to H &E staining. The section is from a medial femoral condyle of a goat,harvested 9 weeks after implantation of the scaffold; D=5.2 mm, L=7.5mm. FIG. 5A is a low magnification of the tissue, while FIG. 5B is ahigher magnification of the insert seen in FIG. 5A. Note the uniformityof the tissue in the region of the hyaline cartilage (inset). There isslight clustering of chondrocytes within the region of regeneration, butthe region otherwise appears comparable to neighboring cartilage tissue.

FIG. 6 depicts the preparation and use of an embodiment of amulti-gradient scaffold of the invention. An embodiment of thepositioning of the plug within the sieve for immersion is shown in FIG.6A. FIGS. 6B and 6C depict light microscopy images of the top portion(panel B) cut from the plug (panel C) and visualized at highermagnification where the size of the voids can be ascertained. FIG. 6Dschematically depicts a multiphasic scaffold embodiment of thisinvention.

FIG. 7 shows micrographs of Safranin O/Fast Green staining of MSCcultures, indicating the chondrogenic potential of the implant as afunction of whether the cells were cultured on Aragonite (FIGS. 7D, 7Eand 7F), Aragonite and Hyaluronic Acid (FIGS. 7G, 7H and 7I) or withoutany scaffold (control) (FIGS. 7A, 7B and 7C) over time.

FIG. 8 shows the stained cells area (FIG. 8A) and intensity (FIG. 8B) ofSafranin O/Fast Green staining of MSCs cultured on Aragonite scaffold,Aragonite and hyaluronic acid and without any scaffold over time.

FIG. 9 shows SEM micrographs of the MSCs, cultured on the variousscaffolds. FIGS. 9A and 9B demonstrate the morphology of MSCs culturedon coral-based scaffolds versus those cultured on coral and Hyaluronicacid-containing scaffolds (FIGS. 9C and 9D).

FIG. 10 shows an embodiment of a scaffold of this inventiondemonstrating a pattern of drilled holes in the bone phase of anembodied implant.

FIGS. 11A-C depict embodiments of scaffolds/implants of this invention.According to this aspect, one phase comprises aragonite and hyaluronicacid 11-10 and another phase comprises aragonite alone 11-20, whereinthe phase further comprises a series of holes or voids along alongitudinal axis 11-30. The terminus of the scaffold according to thisaspect, is tapered 11-40 for ease of insertion as a tight fit, within asite of osteochondral repair. FIG. 11C shows an embodied scaffold,stained with Fast Green, which selectively stains the hyaluronic acidcomponent of the scaffold.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

This invention provides, inter alia, scaffolds, tools and methods of usethereof for repair and/or formation of cartilage and/or bone tissue in asubject. This invention further provides kits for repair and/orformation of cartilage and/or bone tissue in a subject.

Coral, which is comprised of CaCO₃ in the crystalline form of aragoniteor calcite has the advantage of supporting fast cellular invasion,adherence, proliferation and differentiation of mesenchymal stem cellsinto cartilage and/or bone tissue.

Three-dimensional (3-D) coral scaffolds attract mesenchymal stem cellsfrom surrounding or proximally located tissue and promote blood vesselformation to a site of cartilage repair. Such scaffolds can be used forregeneration, repair and enhancement of formation of cartilage and/orbone in a subject for the treatment of full-thickness cartilage defects,partial thickness cartilage defects and/or osteochondral defects.

The terms “coral” and “aragonite” are used interchangeably herein

The coralline-based or calcite-based scaffolds of this invention mayalso be used for regeneration, repair and enhancement of formation ofbone in a subject, for the treatment of a bone condition, disease ordisorder.

This invention provides the unexpected application of coral or calcitescaffolding alone being useful in cartilage and/or bone regeneration,repair and enhancement of formation and moreover, that coral/calcitescaffolding can be prepared and inserted specifically and optimallywithin cartilage and/or bone in a subject in need thereof, for methodsof cartilage and/or bone regeneration, repair and enhancement offormation.

In particular, this invention provides the unexpected application thatcartilage and/or bone regeneration, repair and enhancement of formationis optimal when the coral scaffolding consists essentially of two phaseswherein a first phase of said two phases comprises a coral or biolatticealone and a second phase comprises a coral or biolattice and abiocompatible polymer, wherein the second phase further comprises aseries of hollows along a longitudinal axis in the second phase.

In particular, this invention provides the unexpected advantage in termsof greater chondrogenesis, when the scaffolds as herein describedincorporate hyaluronic acid in a phase inserted within cartilage, andthe underlying pure aragonite containing pre-drilled channels orlongitudinally placed holes allow for greater access of underlyingprogenitor cells throughout the scaffold to stimulate bone/cartilageregeneration and repair, including allowing for greater vascularizationof the implant. The scaffolds of this invention are therefore, in someembodiments, ideally suited for incorporation within a defect site thatspans two different types of tissue, i.e. bone and cartilage.

Scaffolds comprising hyaluronic acid, were shown herein, unexpectedlyand uniquely to promote greater chondrogenesis than scaffolds comprisingthe aragonite alone, and the unique bi-phasic implants of this inventiontake advantage of the improved chondrogenic activity of the phaseincorporating hyaluronic acid, insofar as the phase containinghyaluronic acid is inserted proximally to a cartilage defect site. Thebi-phasic nature of the scaffold as described according to this aspectis uniquely suited therefore to sites spanning both bone and cartilagetissue.

In some embodiments, according to this aspect, the first phase has aheight of between 1-3 mm, or in some embodiments, 0.5-5 mm, or in someembodiments, 1-7 mm. In some embodiments, according to this aspect,hyaluronic acid is distributed throughout the first phase in arelatively homogenous distribution pattern. In some embodiments, a thinlayer of hyaluronic acid may further form above the implant, whichassumes a spongy exterior layer to the implant, at an apical region ofthe implant.

In some embodiments, the biocompatible polymer, such as hyaluronic acidis hydrophilic, and when synovial fluid comes into contact therewith atthe apical layer above the scaffold, or when saline comes into contacttherewith during the implantation procedure, the hyluronic acid absorbsthe fluid and reverts to a hydrogel, as opposed to the pre-implantationdehydrated/dessicated state. This reversion provides mechanicalprotection at the site of implantation, in some embodiments.

In some embodiments, the exterior layer, when “reconstituted” asdescribed following implantation, may elute from the scaffold into thesurrounding site and thereby participate in the stimulation orenhancement of repair at the site, including inter alia, serving as achemoattractant for cells involved in the repair process.

In some embodiments, the invention provides a scaffold for tissuerepair, said scaffold consisting essentially of two phases wherein:

-   -   a first phase of said two phases comprises solid coral or        biolattice comprising hyaluronic acid; and    -   a second phase of said two phases comprises solid coral or        biolattice and said second phase further comprises a series of        hollows along a longitudinal axis in said second phase.

In some embodiments, according to this aspect, the first phase has aheight of between 1-3 mm, or in some embodiments, 0.5-5 mm, or in someembodiments, 1-7 mm. In some embodiments, according to this aspect,hyaluronic acid is distributed substantially homogeneously throughoutthe phase. In some embodiments, a thin layer of hyaluronic acid may formabove the implant, which assumes a spongy exterior layer to the implant,at an apical region of the implant.

According to this aspect, the second phase will contain solid coral orbiolattice, wherein the phase is further modified to incorporate aseries of holes or voids along a longitudinal axis of the phase. In someembodiments, such longitudinal holes may range from 10-60 such holesplaced throughout the phase along a longitudinal axis of the implantaccording to this aspect. In some embodiments, the holes or enlargedvoids will have a diameter ranging from about 250-450 μm. In someembodiments, the holes or enlarged voids will have a diameter rangingfrom about 125-650 μm, or in some embodiments, ranging from about175-550 μm.

According to this aspect, and in some embodiments, the series of holesor voids may be incorporated by physical manipulation of the secondphase, for example, and in some embodiments, solid aragonite or calcitemay be isolated, cleaned and otherwise prepared as described herein, anda drill may be used to create the series of holes/voids as hereindescribed. In some embodiments, other means, such as selectivedissolution of the scaffolding material may be accomplished, where theselective dissolution along a longitudinal axis is accomplished bymethods known in the art, including those described and exemplifiedherein.

According to this aspect, the first phase will contain solid coral orbiolattice, which has not been further modified to alter the porosity ofthe phase, or in some embodiments, may be altered as described furtherhereinunder to specifically alter the pore volume or average porediameter in the phase, whereby such modifications are substantiallyuniform throughout the phase.

Incorporation of a biocompatible polymer such as hyaluronic acid in thefirst phase of the implant may be accomplished via any means, includingpressure-driven application, for example, via application under vacuum,centrifugal force or mechanical pressure. In some embodiments,gravitational force is sufficient to allow appropriate and relativelyhomogenous penetration of the hyaluronic acid to a desired depth of theimplant, creating the first phase as herein described. According to thisaspect, in one embodiment, visual inspection of the implant, for exampleusing the staining with Fast Green/Safranin O, demonstrates uniformdistribution of the hyaluronic acid through the phase and to a desireddepth as a function of the time and conditions of application.

According to this aspect, and in some embodiments, when applying thescaffolds to a site of bone and/or cartilage repair, or in someembodiments, to a defect site where both bone and cartilage are affectedand in need of repair and/or regeneration, the skilled artisan willappreciate that the second phase of the scaffold is inserted within thebone defect site whereas the first phase is inserted within thecartilage defect site.

In some embodiments, such scaffolds may be administered to a subjectwith a bone defect in need of repair, wherein access to the bone defectresults in the creation of a defect in the overlying cartilage, and thescaffolds of this invention allow for the healing of both affectedtissues. In other embodiments, such scaffolds may be administered to asubject with a cartilage defect in need of repair, wherein optimalinsertion of the scaffold for stimulation of cartilage repairnecessitates anchoring of the scaffold in the underlying bone, forexample, by creating a minimal void in the underlying bone for insertionof the scaffold, and once inserted, the scaffold facilitates repair ofboth the overlying cartilage and underlying bone.

In other embodiments, such scaffolds may be administered to a subjectwith an osteochondral defect, where both bone and cartilage tissue arein need of repair as part of the pathogenesis of the disorder. Thescaffolds according to this aspect are, in some embodiments,particularly suited for such applications.

This invention also provides for the unexpected application thatcartilage and/or bone regeneration, repair and enhancement of formationis optimal when the coral scaffolding comprises at least two phases,which phases comprise voids, and vary in terms of the average diameterof the voids within each phase, and/or that cartilage and/or boneregeneration, repair and enhancement of formation is optimal when thecoral scaffolding comprises at least two phases, which phases vary interms of their respective pore volumes (porosity).

It will be appreciated that the term “coral” will refer to a startingmaterial from which aragonite and/or calcite may be isolated.

In one embodiment, the present invention provides a scaffold forinducing or enhancing cartilage or bone regeneration, repair,enhancement of formation, or a combination thereof, which scaffoldconsists of a solid form of aragonite or calcite and further comprises:

-   -   at least a first phase, comprising voids having an average        diameter ranging from about 60-160 μm; and    -   at least a second phase, comprising voids having an average        diameter ranging from about 170-850 μm.

It will be appreciated that according to this aspect, the term “firstphase” and “second phase” do not apply to a particular order withrespect to insertion of the phase within an osteochondral defect, andeither the first phase or the second phase may be oriented to beproximal to cartilage within a repair site, as opposed to the priorembodiment of a scaffold as described hereinabove, wherein the firstphase is inserted proximal to a site of cartilage repair. According tothis aspect, the scaffold may be further modified to comprise both theindicated void average diameter, and either phase may further comprise abiocompatible polymer such as hyaluronic acid and a series of voids orholes along a longitudinal axis of the second phase, as describedhereinabove.

In some embodiments, the term “solid form” with respect to aragonite,refers to solid aragonite harvested from coral, which aragonite istreated to remove debris, proteins and other particulate matter,however, such coral-derived materials are not hydrothermicallytransformed, nor ground, and resuspended.

In some embodiments, the coral for use in the preparation of thescaffolds of this invention may be processed by any means known in theart, for example, as described in PCT International Application SerialNo. PCT/IL2009/000828, which is incorporated by reference as if fullyset forth herein. In some embodiments, the coral may be processedaccording to a process of this invention.

In some embodiments, this invention provides a process for thepurification of a coralline-based scaffolding, said process comprisingthe steps of:

-   -   contacting solid aragonite of a desired size and shape with a        solution comprising an oxidizing agent; and    -   washing and drying said solid aragonite        whereby one or each of said steps is conducted under applied        negative pressure.

According to this aspect, and in some embodiments, the applied negativepressure ranges between about 0.2 to 0.00001 Bar, or in someembodiments, the applied negative pressure ranges between 0.4 to0.0000001 Bar.

According to this aspect, and in some embodiments, the oxidizing agentfor use in the processes of this invention may be any suitable oxidizingagent, which facilitates the removal of organic debris fromcoralline-based scaffolds.

In some embodiments, the oxidizing agent may include, inter alia,potassium nitrate (KNO3), hypochlorite and other hypohalite compounds,iodine and other halogens, chlorite, chlorate, perchlorate, permanganatesalts, ammonium cerium(IV) nitrate, hexavalent chromium compounds,pyridinium chlorochromate (PCC), and chromate/dichromate compounds,peroxide compounds, sulfoxides, persulfuric acid, or nitric acid,acetone, ammonium peroxydisulfate, 1,4-benzoquinone,N-tert-butylbenzensulfinilmidoyl, chloride, tert-butyl hydroperoxide,tert-butyl hypochlorite, 3-chloroperoxybenzoic acid,meta-chloroperbenzoic acid, cumene hydroperoxide, dimethyl sulfoxide,hydrogen peroxide, manganese oxide, meta-chloroperbenzoic acid,N-methylmorpholine-N-oxide, methyltrioxorhenium (MTO), oxalyl chloride,N-tert-butylbenzenesulfinimidoyl chloride, oxone, oxygen, ozone,peracetic acid, periodic acid, peroxy acid, pivaldehyde, potassiumpermanganate, potassium peroxydisulfate, potassium peroximonosulfate,2-propanone, sodium chlorite, sodium percarbonate, sodium periodate,styrene, trichloroisocyanuric acid (TCCA),2,2,6,6-tetramethylpiperidinyloxy TEMPO, tert-butyl hydroperoxide,tert-butyl hypochlorite, tetrabutylammonium peroxydisulphate,trimethylacetaldehyde. In some embodiments, the oxidizing agent issodium hypochlorite.

According to this aspect, and in some embodiments, the process comprisesconducting said contacting under mildly acidic conditions.

According to this aspect, and in some embodiments, the process comprisessubjecting the solid aragonite to a temperature of at least 275° C.under applied negative pressure.

According to this aspect of the invention, the process comprisescontacting the aragonite with an oxidizing agent under applied negativepressure, washing and drying the aragonite applied negative pressure, orboth steps are conducted under applied negative pressure. The appliednegative pressure ranges between 0.2 to 0.00001 Bar, or in someembodiments, between about 0.4 to 0.0000001 Bar, according to thisaspect of the invention.

The scaffolds, kits, processes and methods of this invention make use ofsolid coralline forms.

The solid forms or scaffolds of this invention may be of aragonite orcalcite origin.

In some embodiments, the term “solid form” with respect to calciterefers to calcite isolated from coral, which calcite is treated toremove debris, proteins and other particulate matter, however, suchmaterials are not hydrothermically transformed, nor ground, andresuspended. In some embodiments, the “solid form” calcite refers tocalcite obtained by the preparation of an aragonite solid form, whichform is then converted to calcite by known methods in the art, forexample by exposing the form to high temperature under vacuum.

Any method for conversion of aragonite to calcite as known in the artmay be used to prepare calcite scaffolds of this invention. Example 5presented hereinbelow describes the conversion of aragonite to calciteand demonstrates successful complete transformation thereto (FIG. 6).

The scaffolding of this invention comprises, in some embodiments, aseries of voids, and the at least two phases present in the scaffoldingof this invention vary in terms of the average diameter of the voidspresent in each phase. In some embodiments, the scaffold will compriseat least a first phase, comprising voids having an average diameterranging from about 60-160 μm. In some embodiments, the first phasecomprises voids having an average diameter ranging from about 60-90 μm,or in some embodiments, from about 80-130 μm, or in some embodiments,from about 120-160 0μm.

In some embodiments, the scaffold will comprise at least a second phase,comprising voids having an average diameter ranging from about 170-850μm. In some embodiments, the second phase comprises voids having anaverage diameter ranging from about 170-400 μm, or in some embodiments,from about 250-500 μm, or in some embodiments, from about 450-700 μm orin some embodiments, from about 550-850 μm

In some embodiments, according to this aspect, the scaffold furthercomprises a third phase, comprising voids having an average diameterranging from about 150-300 μm and said second phase comprises voidshaving an average diameter ranging from about 350-850 μm and said thirdphase is positioned between said first and second phase. In someembodiments, such at least third phases may be referred to hereininterchangeably as an “intermediate phase”.

In some embodiments, the scaffold is cylindrical in shape and has adiameter of about 5-15 mm, and a height of about 5-25 mm. In someembodiments, the scaffold has a diameter of about 1-35 mm, and a heightof about 1-45 mm, or about 5-40 mm, and a height of about 5-60 mm, orabout 5-15 mm, and a height of about 5-45 mm.

The average diameter of the voids within the phases of the scaffoldingof this invention may be determined by any means, including digitalimages analysis, as exemplified further hereinbelow. In one embodiment,a coral for use in a scaffold of this invention comprises an averagevoid diameter appropriate for cell seeding and/or development ofvasculature.

The solid forms of this invention comprise at least two phases, whichphases contain pores, owing to the porous nature of the materials ofwhich the scaffolding is comprised. In some embodiments, the phases varyin terms of the pore volume (porosity) of each phase.

In one embodiment, the invention provides a scaffold for the repair ofcartilage, which scaffold consists of a solid form of aragonite orcalcite and further comprises:

-   -   at least a first phase, comprising pores having a pore volume        ranging from about 35-55%; and    -   at least a second phase, comprising pores having a pore volume        ranging from about 56-95%.

It will be appreciated that according to this aspect, the term “firstphase” and “second phase” do not apply to a particular order withrespect to insertion of the phase within a defect site, for example,within an osteochondral defect, and either the first phase or the secondphase may be oriented to be proximal to, for example, the cartilagewithin a repair site, as opposed to the prior embodiment of a scaffoldas described hereinabove, wherein the first phase is inserted proximalto a site of cartilage repair. According to this aspect, the scaffoldmay be further modified to comprise phases comprising pores having theindicated pore volume, and either phase may further comprise abiocompatible polymer such as hyaluronic acid and a series of voids orholes along a longitudinal axis in said second phase, as describedhereinabove.

As used herein, the term “pore volume” refers to volume or open spacesinside the porous scaffolding of this invention. Pore volume isdetermined by any means known in the art. Porosity can be calculated bystandard methods, an example of which is provided further hereinbelow,see for example, Karageorgiou V, Kaplan D. (2005) “Porosity of 3Dbiomaterial scaffolds and osteogenesis” Biomaterials.; 26(27):5474-91,which is hereby incorporated by reference in its entirety.

In some embodiments, according to this aspect, the scaffold comprises atleast a first phase, comprising pores having a pore volume ranging fromabout 35-45% and in some embodiments the scaffold comprises a firstphase comprising pores having a pore volume ranging from about 40-55%.

In some embodiments, according to this aspect, the scaffold comprises atleast a second phase, comprising pores having a pore volume ranging fromabout 56-70% and in some embodiments, ranging from about 60-74% or thescaffold comprises a second phase comprising pores having a pore volumeranging from about 65-75%, or in some embodiments, ranging from about70-85%, or in some embodiments, ranging from about 80-95%.

In some embodiments, according to this aspect, the scaffold furthercomprises a third phase, comprising pores having a pore volume rangingfrom about 80-95%, wherein said second phase comprises voids having anaverage diameter ranging from about 56-85% and said third phase ispositioned between said first and second phase.

In one, embodiment, the term “about” refers to a variance of from 1-10%,or in another embodiment, 5-15%, or in another embodiment, up to 10%, orin another embodiment, up to 25% variance from the indicated values,except where context indicates that the variance should not result in avalue exceeding 100%.

In some embodiments, the invention provides a scaffold for the repair,regeneration or enhancement of formation of cartilage, bone, or acombination thereof, which scaffold consists of a solid form ofaragonite or calcite isolated from a coral and further comprises:

-   -   at least a first phase, comprising voids having an average        diameter, pore volume or combination thereof, which corresponds        to that of the native coral from which said solid form was        isolated; and    -   at least a second phase, comprising voids having an average        diameter, pore volume or combination thereof, which average void        diameter, pore volume or combination thereof is greater than        that of said first phase by from about 15-100%.

It will be appreciated that according to this aspect, the term “firstphase” and “second phase” do not apply to a particular order withrespect to insertion of the phase within a defect site, for example,within an osteochondral defect, and either the first phase or the secondphase may be oriented to be proximal to, for example, the cartilagewithin a repair site, as opposed to the prior embodiment of a scaffoldas described hereinabove, wherein the first phase is inserted proximalto a site of cartilage repair. According to this aspect, the scaffoldmay be further modified to comprise phases comprising pores having theindicated pore volume, or voids having the indicated average diameter,or combinations thereof and either phase may further comprise abiocompatible polymer such as hyaluronic acid and a series of voids orholes along a longitudinal axis in the second phase.

In some embodiments, according to this aspect, the scaffold comprisesvoids having an average diameter, pore volume or combination thereof,which average pore diameter, pore volume or combination thereof isgreater than that of said first phase by from about 15-35% and in someembodiments, ranging from about 60-74%, or the scaffold comprises asecond phase comprising pores having a pore volume ranging from about45-65%, or in some embodiments, ranging from about 50-85%, or in someembodiments, ranging from about 80-95%.

In some embodiments, the invention provides a scaffold for the repair,regeneration or enhancement of formation of cartilage, bone, or acombination thereof, which scaffold consists of a solid form ofaragonite or calcite isolated from a coral and further comprises:

-   -   at least a first phase, comprising voids having an average        diameter, pore volume or combination thereof, which corresponds        to that of the native coral froth which said solid form was        isolated; and    -   at least a second phase, comprising voids having an average        diameter, pore volume or combination thereof, which average void        diameter, pore volume or combination thereof is greater than        that of said first phase by from about 15-900%.

It will be appreciated that according to this aspect, the term “firstphase” and “second phase” do not apply to a particular order withrespect to insertion of the phase within a defect site, for example,within an osteochondral defect, and either the first phase or the secondphase may be oriented to be proximal to, for example, the cartilagewithin a repair site, as opposed to the prior embodiment of a scaffoldas described hereinabove, wherein the first phase is inserted proximalto a site of cartilage repair. According to this aspect, the scaffoldmay be further modified to comprise phases comprising pores having theindicated pore volume, or voids having the indicated average diameter,or combinations thereof and either phase may further comprise abiocompatible polymer such as hyaluronic acid and a series of voids orholes along a longitudinal axis of said phase, wherein the biocompatiblepolymer such as hyaluronic acid is located substantially within suchseries of voids or holes.

In some embodiments, according to this aspect, the scaffold comprises asecond and third phase having voids having an average diameter, porevolume or combination thereof, which average pore diameter, pore volumeor combination thereof is greater than that of said first phase by fromabout from about 15-900% wherein, in some embodiments, the third phasecomprises voids having an average diameter, pore volume or combinationthereof ranging from about 300-900%, or in some embodiments, 300-450%,or in some embodiments, 425-600%, or in some embodiments, 575-900% andthe scaffold comprises a second phase comprises voids having an averagediameter, pore volume or combination thereof ranging from about rangingfrom about 15-200%, or in some embodiments, 50-125%, or in someembodiments, 125-200%, of that which is in the first phase.

According to this aspect, and in on embodiment, the scaffold willcomprise a third phase, comprising voids having an average diameter,pore volume or combination thereof, which average pore diameter, porevolume or combination thereof is greater than that of said first phaseby from about 15-35% and said second phase comprises voids having anaverage diameter, pore volume or combination thereof, which average porediameter, pore volume or combination thereof is greater than that ofsaid first phase by from about 40-100% and said third phase ispositioned between said first and second phase.

It will be appreciated that different species of coral vary in terms oftheir average pore diameter and pore volume and the inventioncontemplates use of any such coral as a starting material for thepreparation of the scaffolds as herein described, where the scaffold ischaracterized in that it possesses at least two phases, wherein a firstphase contains voids, and a pore volume native to the coral from whichthe scaffolds are prepared, and a second phase, whose voids areenlarged, whose overall pore volume increases, or a combination thereof.

In one embodiment, this invention provides a scaffold for repair ofcartilage comprising a biolattice consisting essentially of calcitecapable of being inserted within a site of cartilage repair. In someembodiments, the biolattice is derived from Tetraclita rufotincta.

The term biolattice refers to a CaCO3-containing biomaterial which iscrystalline or amorphous and derived from, inter alia, a coral orbarnacle species.

Calcite polymorphs of calcium carbonate from natural limestone have beendescribed {Fujita Y, Yamamuro T, Nakamura T, Kotani S, Ohtsuki C, KokuboT. J Bimed Mater Res. 1991 August; 25(8):991-1003}. In vitrotransformation of calcite by heating aragonite isolated from naturalcoral, has been described as well (Fricain J C, Bareille R, Ulysse F,Dupuy B, Amedee J. J. Biomed Mater Res. 1998 October; 42(1):96-102);

In another embodiment, this invention provides a scaffold for tissuerepair, said scaffold comprising at least two phases wherein a firstphase of said two phases comprises a coral or biolattice and a secondphase comprises a biocompatible polymer or polymers.

In one embodiment, the term “proximal” refers to something beingsituated close to a particular locale. In one embodiment, a scaffold ofthis invention is forcibly held in position within a site of cartilagerepair by a raised region of the scaffold contacting tissue situated ator proximal to a site of cartilage repair.

By optimizing the specific positioning of a scaffold the porouscrystalline structure of a coral scaffolds of this invention, describedbelow, is accessible to beneficial components located within a tissuemilieu. For example, the porous crystalline structure of coral allowsin-growth of blood vessels to create a blood supply for the cartilagethat will infiltrate the scaffold during cartilage repair. Bypenetrating into a bone marrow void, mesenchymal stem cells locatedwithin the bone marrow now have access to the exposed surface of thescaffold. In one embodiment, the region of the scaffold penetrating intoa bone marrow void attracts mesenchymal stem cells from the bone marrowand promotes blood vessel formation to the site of cartilage repair. Inone embodiment, the region of the scaffold penetrating into a bonemarrow void promotes adhesion, proliferation, or differentiation or acombination thereof, of the mesenchymal stem cells attracted to thescaffold.

Thus, it will be apparent to one skilled in the art that the specificpositioning of the scaffold within a site of cartilage repair arrangesthe scaffold of this invention such that the scaffold is most effectivefor cartilage repair.

In some embodiments, the region of the scaffold which penetrates throughbone and stably inserts within bone marrow is also the region of thescaffold which positions and confines the scaffold within a site ofcartilage repair, or in some embodiments, the region of the scaffoldwhich penetrates through bone and stably inserts within bone marrow isnot the region which positions and confines the scaffold within a siteof cartilage repair. In one embodiment, the region inserts in such a waythat no other portion of the scaffold is in contact with tissue at thesite. In another embodiment, the region inserts in such a way that theside walls of the scaffold make contact with tissue at the site ofcartilage repair.

In some embodiments, the scaffold is of a shape which accommodates asite of repair.

In some embodiments, the scaffold approximates the form of a cylinder,cone, tac, pin, screw, rectangular bar, plate, disc, pyramid, granule,ball or cube.

In some embodiments, the scaffolds of this invention may be used inconjunction with other known and/or available materials forstimulating/enhancing bone and/or cartilage repair. In some embodiments,the scaffolds of this invention may be utilized to affix additionalscaffolds, for example for use in whole joint repair or ligament repair,or other connector tissue repair.

In some embodiments, the scaffolds of this invention may be used forexample, as a pin, in conjunction with other scaffolds for bone repairor regeneration, etc. It is to be understood that any use of thescaffolds of this invention, alone or in conjunction with otherappropriate materials, for the treatment, repair or stimulation ofgrowth of bone and/or cartilage is to be considered as part of thisinvention

It will be appreciated that the scaffolds of this invention may be ofany suitable shape or size to accommodate its application in accordancewith the methods of this invention. For example, and in someembodiments, for applications of the scaffolds of this invention withinlong bones of a subject, the dimensions of the scaffold will be scaledto approximate that of the site into which the scaffold will beimplanted, and may be on an order of magnitude scaling from millimetersto centimeters, as needed. Similarly, shapes of the scaffolds of theinvention may be any shape into which the scaffolds of this inventionmay be machined or processed, and may have any configuration as will beappropriate to achieve the desired growth, repair or regeneration ofbone and/or cartilage.

In some embodiments, the scaffold comprises a hollow or hollows along aCartesian coordinate axis of said scaffold and in some embodiments, theaxis is a long axis of said scaffold.

In some embodiments, the invention provides a kit for the repair,regeneration or enhancement of formation of cartilage, bone, or acombination thereof comprising the scaffold of this invention,directions for utilizing said scaffold in the repair, regeneration orenhancement of formation of cartilage, bone, or a combination thereofand optionally a tool or tools for optimal insertion of said scaffold,seeding said scaffold with cells or a combination thereof.

In one embodiment, the coral is seeded with a precursor cell. In oneembodiment, the precursor cell is a mesenchymal stem cell. In otherembodiments, the cell may be a mesenchymal cell; chondrocyte;fibrochondrocyte; osteocyte; osteoblast; osteoclast; synoviocyte; bonemarrow cell; stromal cell; stem cell; embryonic stem cell; precursorcell, derived from adipose tissue; peripheral blood progenitor cell;stem cell isolated from adult tissue; genetically transformed cell; or acombination thereof. In another embodiment, a precursor cell may referto a combination of chondrocytes and other cells; a combination ofosteocytes and other cells; a combination of synoviocytes and othercells; a combination of bone marrow cells and other cells; a combinationof mesenchymal cells and other cells; a combination of stromal cells andother cells; a combination of stem cells and other cells; a combinationof embryonic stem cells and other cells; a combination of precursorcells isolated from adult tissue and other cells; a combination ofperipheral blood progenitor cells and other cells; a combination of stemcells isolated from adult tissue and other cells; and a combination ofgenetically transformed cells and other cells. In one embodiment of thepresent invention; the precursor cells for use in the method of thepresent invention are prepared from an organ tissue of the recipientmammal (i.e. autologous), or a syngeneic mammal. In another embodiment,allogeneic and xenogeneic precursor cells may be utilized.

In some embodiments, the scaffold comprises a third phase comprising acoral which differs in composition from said first phase.

In some embodiments, the third phase is positioned between said firstphase and said second phase.

In some embodiments, the third phase is positioned proximally to saidfirst phase and distally to said second phase.

In some embodiments, the third phase is positioned proximally to saidsecond phase and distally to said first phase.

In some embodiments, the first phase or said second phase is insertedinto a region which is proximal to subchondral bone.

In some embodiments, the scaffold may comprise a third phase, which maybe inserted into a region which is proximal to subchondral bone.

In some embodiments, the phase which is inserted comprises at least aterminal modification, which enhance the tissue repair

In one embodiment, a site of cartilage repair may be considered tocomprise a 3 dimensional (3-D) space at or proximal to a site of acartilage and/or defect or potential defect. In one embodiment, this 3-Dspace comprises at least a wall or a floor, or a combination thereof,and positioning within such a site may be described herein, relative tosaid wall or floor, or in some embodiments, positioning may be relativeto insertion within a tissue site proximal to said wall or floor. Insome embodiments, positioning include insertion of the scaffold or aregion thereof, past the wall and/or floor of cartilage and/or bonetissue or a site of defect or injury or potential defect or injury inthe cartilage and/or bone tissue, such that insertion into bone tissueoccurs.

One skilled in the art will recognize that the shape of a site ofcartilage and/or bone repair and the shape of a 3-D scaffold of thisinvention provide many different combinations for stably positioning ascaffold within a site of cartilage repair and/or bone. In oneembodiment, a scaffold of this invention is shaped prior to use inmethods of this invention for cartilage repair and/or bone. In oneembodiment, a scaffold of this invention is shaped concurrent to use inmethods of this invention for cartilage and/or bone repair. By shaping ascaffold concurrent with use of the scaffold in methods of thisinvention, the dimensions of the scaffold may be precisely selected forspecific positioning of the scaffold within a site of repair. It will beappreciated that multiple scaffolds of this invention may be placedwithin or shaped and placed within a site of cartilage and/or bonerepair.

In some embodiments, reference to a “scaffold”, “implant” or “plug”, asused herein refers to any embodiment or combined embodiments as hereindescribed with regard to the scaffolds to be considered as beingincluded in the described aspect of this invention. For example,reference to a “scaffold” as used herein, is to be understood to referto any embodiment of a scaffold as described herein being applicable forthe indicated purpose or containing the indicated attribute, etc.

In one embodiment, “scaffold” refers to a shaped platform used forcartilage and/or bone repair, wherein the shaped platform provides asite for cartilage and/or bone regeneration. In one embodiment, thescaffold is a temporary platform. In one embodiment, “temporaryplatform” refers to a natural degradation of a coral of this inventionthat occurs over time during cartilage and/or bone repair, wherein thenatural fully or partially degradation of the coral may results in achange of scaffold shape over time and/or change in scaffold size overtime.

In one embodiment, the coral is shaped in the form of the tissue to begrown. For example, the coral can be shaped as a piece of cartilaginoustissue, such as a meniscus for a knee or elbow; a joint; an articularsurface of a bone, the rib cage, a hip, a pelvis, an ear, a nose, aligament, the bronchial tubes and the intervertebral discs.

This invention provides, in some embodiments, coral scaffolds for use inrepairing cartilage and/or bone tissue defects associated with physicaltrauma, or cartilage and/or bone tissue defects associated with adisease or disorder in a subject.

In one embodiment of this invention, the term “coral” refers to coralwhich is cut from a single piece of coral. In one embodiment, the coralhas pore-like cavities or interstices.

In one embodiment, the coral scaffold is shaped prior to use in a methodof cartilage and/or bone repair. In one embodiment, the coral scaffoldis shaped concurrent with a method of cartilage and/or bone repair,e.g., the coral scaffold may be shaped during surgery when the site ofrepair may be best observed, thus optimizing the shape of the scaffoldused.

In one embodiment, the scaffolds, methods and/or kits of this inventionemploy use of a coral. In one embodiment, the coral comprise anyspecies, including, inter alia, Porites, Acropora, Millepora, or acombination thereof.

In one embodiment, the coral is from the Porites species. In oneembodiment, the coral is Porites Lutea. In most species, the void tosolid ratios is generally in the range of 0.4 to 0.6, and the void phasecompletely interconnects, forming a highly regular network thatinterpenetrates the solid calcium carbonate phase. In one embodiment,this uniform and interconnecting architecture is particularly useful asa framework in the scaffolds, methods and/or kits of this invention.

In one embodiment, the coral is from the Acropora species. In oneembodiment, the coral is Acropora grandis, which in one embodiment isvery common, fast growing, and easy to grow in culture. Thus, in oneembodiment Acropora samples can be easily collected in sheltered areasof the coral reefs and collection from the coral reefs can be avoided byuse of cultured coral material.

The average skeletal density of Acropora grandis is 2.7 g/ml. Becausethe skeleton of this coral species is dense and strong, it can be easilymachined to a variety of configurations of shaped products or structuresof different sizes, for example by grinding. This material isparticularly suited for use in an implant device, in particular forweight-bearing joints such as knee and hip joints, where strength is anessential property of the implant device. Thus, in one embodiment,Acropora coral is useful as a framework in the scaffolds, methods and/orkits of this invention.

In another embodiment, the coral is from the Millepora species. In oneembodiment, the coral is Millepora dichotoma. In one embodiment, thecoral has a pore size of 150 μm and can be cloned and cultured, makingMillepora useful as a framework in the scaffolds, methods and/or kits ofthis invention.

In another embodiment, the coral is from any one or more of thefollowing species: Favites halicora; Goniastrea retiformis; Acanthastreaechinata; Acanthastrea hemprichi; Acanthastrea ishigakiensis; Acroporaaspera; Acropora austera; Acropora sp. “brown digitate”; Acroporacarduus; Acropora cerealis; Acropora chesterfieldensis; Acroporaclathrata; Acropora cophodactyla; Acropora sp. “danai-like”; Acroporadivaricata; Acropora donei; Acropora echinata; Acropora efflorescens;Acropora gemmifera; Acropora globiceps; Acropora granulosa; Acropora cfhemprichi; Acropora kosurini; Acropora cf loisettae; Acroporalongicyathus; Acropora loripes; Acropora cf lutkeni; Acroporapaniculata; Acropora proximalis; Acropora rudis; Acropora selago;Acropora solitaryensis; Acropora cf spicifera as per Veron; Acropora cfspicifera as per Wallace; Acropora tenuis; Acropora valenciennesi;Acropora vaughani; Acropora vermiculata; Astreopora gracilis; Astreoporamyriophthalma; Astreopora randalli; Astreopora suggesta; Australomussarowleyensis; Coscinaraea collumna; Coscinaraea crassa; Cynarinalacrymalis; Distichopora violacea; Echinophyllia echinata; Echinophylliacf echinoporoides; Echinopora gemmacea; Echinopora hirsutissima;Euphyllia ancora; Euphyllia divisa; Euphyllia yaeyamensis; Faviarotundata; Favia truncatus; Favites acuticollis; Favities pentagona;Fungia granulosa; Fungia klunzingeri; Fungia mollucensis; Galaxeaacrhelia; Goniastrea edwardsi; Goniastea minuta; Hydnophora pilosa;Leptoseris explanata; Leptoseris incrustans; Leptoseris mycetoseroides;Leptoseris scabra; Leptoseris yabei; Lithophyllon undulatum; Lobophylliahemprichii; Merulina scabricula; Millepora dichotoma; Millepora exaesa;Millipora intricata; Millepora murrayensis; Millipora platyphylla;Monastrea curta; Monastrea colemani; Montipora caliculata; Montiporacapitata; Montipora foveolata; Montipora meandrina; Montiporatuberculosa; Montipora cf vietnamensis; Oulophyllia laevis; Oxyporacrassispinosa; Oxypora latera; Pavona bipartita; Pavona venosa; Pectiniaalcicornis; Pectinia paeonea; Platygyra acuta; Platygyra pini; Platygyrasp “green”; Platygyra verweyi; Podabacia cf lanakensis; Porites annae;Porites cylindrica; Porites evermanni; Porites monticulosa; Psammocoradigitata; Psammocora explanulata; Psammocora haimeana; Psammocorasuperficialis; Sandalolitha dentata; Seriatopora caliendrum;Stylocoeniella armata; Stylocoeniella guentheri; Stylaster sp.; Tubiporamusica; Turbinaria stellulata; or any coral known in the art, or acombination thereof.

In another embodiment, coral for use in the scaffolds, methods and/orkits of this invention may be Madreporaria, Helioporida of the orderCoenothecalia, Tubipora of the order Stolonifera, Millepora of the orderMilleporina, or others known in the art. In some embodiments, coral foruse in the scaffolds, methods and/or kits of this invention may comprisescleractinian coral, including in some embodiments, Goniopora andothers. In some embodiments, coral for use in the scaffolds, methodsand/or kits of this invention may comprise Alveoppora. In someembodiments, coral for use in the scaffolds, methods and/or kits of thisinvention may comprise bamboo corals, including in some embodiments,coral from the family Isididae, genera Keratoisis, Isidella, and others.

As described above, a scaffold's region's ability to position andconfine the scaffold of this invention is dependent on the region'sgeometry and the geometry at the site of cartilage and/or bone repairwhere the scaffold will be implanted. In one embodiment, the region'sgeometry comprises a sharp edge. In one embodiment, the region'sgeometry comprises a rounded edge. In one embodiment, the region'sgeometry comprises a jagged edge.

In one embodiment of this invention, an optimal depth and angle within asite of cartilage and/or bone repair comprise the depth and angle mostbeneficial for cartilage and/or bone repair. In one embodiment, theoptimal depth and angle most beneficial comprise a position so that ascaffold of this invention is accessible to a pool of mesenchymal stemcells, a tissue milieu, blood vessels, nutrients, an effector compound,or a therapeutic compound, or a combination thereof.

In one embodiment of this invention, the term “depth” refers to ameasurement of a scaffold of this invention extending from an imaginaryline resting on the open surface of a repair site to a place beneath thetissue floor at a site of cartilage and/or bone repair.

It will be recognized by one skilled in the art that the depth of otherregions of the scaffold may not be below any tissue surface. Forexample, and in an embodiment of this invention, based on a site ofcartilage repair shaped like a cylindrical pit, an imaginary line drawnto rest across the opening of the pit represents the top of the pit. Inone embodiment, positioning of the scaffold results in the entirety ofthe scaffold being below the top of the pit and therefore at a depthbelow the imaginary line across the opening. In one embodiment,positioning of the scaffold results in a portion of the scaffold beingabove the top of the pit and therefore not wholly within a site ofcartilage repair. The benefit of placing a scaffold at a given depth maydepend on the resulting contact the scaffold makes with surroundingtissue, either within the site of cartilage repair or proximal to thesite of cartilage repair.

Similarly, and in another aspect of the invention, with regard toimplantation of the scaffolds of this invention within bone, the site ofimplantation may as well be envisioned as a pit, with an imaginary linedrawn to rest across the opening of the pit, representing the top of thepit. According to, this aspect, positioning of the scaffold results inthe entirety of the scaffold being below the top of the pit or in someembodiments, positioning of the scaffold may result in a portion of thescaffold being above the top of the pit and therefore not wholly withinthe site of bone repair. The benefit of placing a scaffold at a givendepth may depend on the resulting contact the scaffold makes withsurrounding tissue, either within the site of bone repair or proximal tothe site of bone repair.

In one embodiment, the term “angle” refers to a measurement of the arcformed by an imaginary line along the long axis of the scaffold and animaginary plumb line perpendicular to the line resting at the opening ofa site of cartilage and/or bone repair described above, with the arcprogressing in a clockwise direction around this imaginary plumb line.Thus, in one embodiment a Scaffold of this invention may be positionedand confined at an optimal depth and angle such that the scaffold isparallel to the perpendicular line, and therefore the angle would be 0degrees. In one embodiment a scaffold of this invention may bepositioned perpendicular to the imaginary plumb line, and therefore theangle would be 90 degrees. In one embodiment, the scaffold ispositioned, and confined at an angle equaling or less than 10 degrees.In one embodiment, the scaffold is positioned and confined at an angleequaling or less than 35 degrees. In one embodiment, the scaffold ispositioned and confined at an angle equaling or less than 55 degrees. Inone embodiment, the scaffold is positioned and confined at an angleequaling or less than 75 degrees. In one embodiment, the scaffold ispositioned and confined at an angle equaling or less than 95 degrees. Inone embodiment, the scaffold is positioned and confined at an angleequaling or less than 115 degrees. In one embodiment, the scaffold ispositioned and confined at an angle equaling or less than 125 degrees.In one embodiment, the scaffold is positioned and confined at an angleof less than 145 degrees. In one embodiment, the scaffold is positionedand confined at an angle equaling or less than 165 degrees. In oneembodiment, the scaffold is positioned and confined at an angle lessthan 180 degrees

In some embodiments, multiple scaffolds are inserted to maximally occupya defect site, such that each scaffold material may be inserted at adifferent angle and/or shape and/or depth and/or porosity, toaccommodate proper insertion into the desired region within a site ofcartilage and/or bone repair. It is to be understood that the referenceto angles of positioning above may be with regard to one or morescaffolds inserted in a particular cartilage and/or bone defect site.

Contact between exposed surfaces of a scaffold and tissue at or proximalto a site of cartilage and/or bone repair provides a bioactive surfacewhich, in the methods of use of this invention may include or enhancecartilage and/or bone repair. For example, in one embodiment, theexposed surface of a scaffold provides a bioactive surface attractingmesenchymal stem cells. In another embodiment, the exposed surfaceprovides a place for mesenchymal stem cell attachment, growth,proliferation, or differentiation, or a combination thereof, allprocesses which induce or enhance cartilage repair. In addition, theexposed surface of a scaffold may attract blood vessels. Moreover,tissue at or proximal to a site of cartilage and/or bone repair may be arich source of nutrients, effector compounds, therapeutic compounds, ora combination thereof, which may be beneficial in cartilage and/or bonerepair so that contact between an exposed surface of a scaffold and suchtissue induces or enhances cartilage and/or bone repair.

In one embodiment, the angle of placement of a scaffold is such that thescaffold is in contact with a region of a wall within a site ofcartilage and/or bone repair. In one embodiment, a scaffold of thisinvention may be positioned and confined such that there is maximalcontact between the scaffold and tissues at or proximal to a site ofcartilage and/or bone repair. In one embodiment, a scaffold of thisinvention may be positioned and confined such that a region of thescaffold penetrates a subchondral bone and/or bone marrow void and thereis maximal contact between the scaffold and tissues at or proximal to asite of cartilage and/or bone repair. In one embodiment, contact betweenthe exposed surface of the scaffold and the tissue at or proximal to asite of cartilage and/or bone repair provides maximal surface area ofthe scaffold for interaction with a population of mesenchymal stemcells, blood vessels, effector compounds, or other components of atissue milieu, or a combination thereof.

In one embodiment, placing and confining a scaffold of this invention atan optimal depth and angle within a site of cartilage and/or bone repairprovides for penetration of a portion of the exposed surface of thescaffold, through a bone tissue.

By optimizing the specific positioning of a scaffold the porouscrystalline structure of the scaffolds of this invention, describedbelow, is accessible to beneficial components located within a tissuemilieu For example, the porous crystalline structure of the scaffoldallows in-growth of blood vessels to create a blood supply for thecartilage and/or bone that will infiltrate the scaffold during cartilageand/or bone repair. In one embodiment, the scaffold attracts mesenchymalstem cells and promotes blood vessel formation to the site of cartilagerepair.

Thus, it will be apparent to one skilled in the art that the specificpositioning of the scaffold within a site of cartilage and/or bonerepair arranges the scaffold of this invention such that the scaffold ismost effective for cartilage and/or bone repair.

In one embodiment, “scaffold” refers to a shaped platform used forcartilage and/or bone repair, wherein the shaped platform provides asite for cartilage and/or bone formation and/or regeneration. In oneembodiment, the scaffold is a temporary platform. In one embodiment,“temporary platform” refers to a natural fully or partially degradationof a coral of this invention that occurs over time during cartilagerepair, wherein the natural degradation of the coral may results in achange of scaffold shape over time and/or a change in scaffold size overtime.

In one embodiment, the coral is shaped in the form of the tissue to begrown. For example, the coral can be shaped as a piece of cartilaginousor bony tissue, such as a meniscus for a knee or elbow; a joint; anarticular surface of a bone, the rib cage, a hip, a pelvis, an ear, anose, the bronchial tubes, the intervertebral discs, a ligament, avertebra, the tibia, the femur, the shoulder and the jaw.

This invention provides, in some embodiments, coral scaffolds for use inrepairing cartilage and/or bone tissue defects associated with physicaltrauma, or cartilage and/or bone tissue defects associated with adisease or disorder in a subject.

In one embodiment of this invention, the term “coral” refers to coralwhich is cut from a single piece of coral. In one embodiment; the coralhas pore-like cavities or interstices.

In one embodiment, the coral scaffold is shaped prior to use in a methodof cartilage and/or bone repair. In one embodiment, the coral scaffoldis shaped concurrent with a method of cartilage and/or bone repair,e.g., the coral scaffold may be shaped during surgery when the site ofrepair may be best observed, thus optimizing the shape of the scaffoldused.

In one embodiment, the size of a scaffold may be any size that would beuseful for the purposes of the Present invention, as would be known toone skilled in the art. In one embodiment, the scaffold or a portionthereof may be about the size of a site of cartilage and/or bone repair.In one embodiment, the scaffold or a portion thereof may be about thesize of a cartilage and/or bone defect so that the scaffold may beplaced within a site of cartilage and/or bone repair. In anotherembodiment, the scaffold may be larger than the size of a cartilageand/or bone defect. For example, in one embodiment, the scaffold of thisinvention may be larger than the size of a cartilage and/or bone defect,whereby the scaffold may extend to a site of mesenchymal cellavailability. In one embodiment, the scaffold may be smaller than thesize of a cartilage and/or bone defect.

In some embodiments, the scaffold size will be on a millimeter scale,for example, having at least one long axis of about 2-200 mm, or in someembodiments, about 1-18 mm, or in some embodiments, about 0.5 mm-3 mm,or in some embodiments, about 6-12 mm, or in some embodiments, about10-15 mm, or in some embodiments, about 12-40 mm, or in someembodiments, about 30-100 mm, or in some embodiments, about 50-150 mm,or in some embodiments, about 100-200 mm.

In some embodiments the scaffold size will be on the centimeter scale,for example having at least one long axis of about 0.5-30 cm

In one embodiment, the scaffold may be about the same size as a tissuevoid at a site of tissue repair. This tissue void may be due to acartilage and/or bone defect, cartilage and/or bone degeneration or mayhave been created artificially during methods of cartilage and/or bonerepair or any combination thereof. In one embodiment, the tissue voidcomprises an absence of cartilage and/or bone tissue. In one embodiment,the scaffold or a portion thereof may be the size of a cartilage and/orbone defect such that the scaffold may be placed within a site ofcartilage and/or bone repair to enhance cartilage and/or bone formationat the site of cartilage and/or bone repair. In another embodiment, thescaffold may be larger than the size of a cartilage and/or bone defectso that the scaffold may reach to a site of mesenchymal stem cellavailability.

In some embodiments, a tight fit is desirable with regard to fitting theimplant within the site of tissue repair. According to this aspect, andin some embodiments, it may be desirable to taper a terminus of thescaffolds of this invention for easy insertion within a tight space foroptimal tight fitting of the implant. FIG. 11 for example, shows aschematic of an embodied scaffold of this invention, whereby theterminus inserting into bone, in the second phase of the scaffold istapered (11-40) to accommodate an easier tight fit.

In one embodiment of this invention, “about” refers to a quality whereinthe means to satisfy a specific need is met, e.g., the size may belargely but not wholly that which is specified but it meets the specificneed of cartilage and/or, bone repair at a site of cartilage and/or bonerepair. In one embodiment, “about” refers to being closely orapproximate to, but not exactly. A small margin of error is present.This margin of error would not exceed plus or minus the same integervalue. For instance, about 0.1 micrometers would mean no lower than 0but no higher than 0.2.

In one embodiment, the term “void” refers to a space not occupied. Inthe instant invention, for example, in one embodiment, a void may be aspace in a scaffold naturally not occupied. In one embodiment, a voidmay be a space not occupied at a site of repair. In one embodiment, avoid may be a space not occupied within a scaffold of the currentinvention. In one embodiment, a void may be a volume of a pore or a poreregion.

In one embodiment, coral is washed, bleached, frozen, dried, sterilizedor a combination thereof. In some embodiments, the coral is processed asexemplified further hereinunder. In some embodiments, the coral, onceprocessed into the scaffolds of this invention are seeded with a desiredpopulation of cells or populations of cells, prior to implantationwithin a site of cartilage and/or bone repair.

In one embodiment, this invention provides a process for the preparationof a multi-phasic scaffold for the repair of cartilage, said processcomprising the steps of:

-   -   contacting only a portion of a solid form of aragonite or        calcite with a calcium chelator and an acid to yield a solid        form comprising enlarged voids in at least a portion of said        solid form; and    -   washing and drying said solid form under applied negative        pressure.

In some embodiments, the calcium chelator is EDTA. In one embodiment,the chelator may comprise: ethylenediamine-N,N,N′,N′-tetraacetic acid(EDTA),O,O′-bis(2-aminophenylethyleneglycol)ethylenediamine-N,N,N′,N′-tetraaceticacid (BAPTA), N,N-bis(2-hydroxyethyl)glycine (Bicine),trans-1,2-diaminocyclohexane-ethylenediamine-N,N,N′,N′-tetraacetic acid(CyDTA),1,3-diamino-2-hydroxypropane-ethylenediamine-N,N,N′,N′-tetraacetic acid(DPTA-OH), diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (DPTA),ethylenediamine-N,N′-dipropionic acid dihydrochloride (EDDP),ethylenediamine-N,N′-bis(methylenephosphonic acid) hemihydrate (EDDPO),N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid (EDTA-OH),ethylenediamine-N,N,N′,N′-tetrakis(methylenephosphonic acid) (EDTPO),O,O′-bis(2-aminoethyl)ethyleneglycol tetraacetic acid (EGTA),N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED),1,6-hexamethylenediamine-N,N,N′,N′-tetraacetic acid (HDTA),N-(2-hydroxyethyl)iminodiacetic acid (HIDA), iminodiacetic acid (IDA),1,2-diaminopropane-N,N,N′,N′-tetraacetic acid (methyl-EDTA),nitrilotriacetic acid (NTA), nitrilotripropionic acid (NTP),nitrilotris(methylenephosphonic acid) trisodium salt (NTPO),N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), andtriethylenetetramine-N,N,N′,N″,N″-hexaacetic acid (TTHA), rhod-2, DMSA,FLUO 3, FURA 2, INDO 1, QUIN 2, or other chelators known in the art, ora combination thereof.

In some embodiments, the acid is formic acid. In some embodiments, theacid is a weak acid, such as picric acid, acetic acid, or others knownto the skilled artisan. In some embodiments, the acid is a strong acidsuch as hydrochloric acid, nitric acid, sulfuric acid, or others, knownto the skilled artisan. In some embodiments, the acid is a hydrogenhalide, halogen oxoacid, such as: hypochloric acid, chloric acid,perchloric acid, periodic acid a fluorosulfuric acid, a nitric acid, aphosphoric acid, a fluoroantimonic acid, a fluoroboric acid, ahexafluorophosphoric acid, acetic acid, citric acid, gluconic acid,lactic acid, oxalic acid, tartaric acid or a chromic acid.

Without being bound by theory, the processes of this invention make useof calcium chelator, which acts as a slow decalcificator of thecoralline material. The chelator, for example, EDTA, binds ionizedcalcium present on the outer layer of the mineral crystal, slowlyreducing the size of the crystal. Addition of a chelator alone may insome embodiments, be sufficient to arrive at the scaffolds of thisinvention.

In accordance with this aspect, the coralline material is furthercontacted with an acid, for example, formic acid. Without being bound bytheory, the addition of the acid results in faster dissolution of thecoralline material, as compared to samples contacted with a chelatoralone.

In some embodiments, the combined application of chelator and acidresults in a controlled dissolution, providing for a homogenous poresize and volume.

In some embodiments, the choice of chelator, or acid, the concentrationof each, or a combination thereof will provide for additional control ofthe enlarged voids within the thereby produced coralline-based scaffoldsof this invention. It will be appreciated that the artisan will pick aweak or strong acid, at high or low concentration, and favor certaincalcium chelators to arrive at a desired pore volume or average diameterfor the voids enlarged in the scaffolds as herein described, produced bythe methods of this, invention and such choice is to be considered anembodied aspect of the processes of this invention. For example, and insome embodiments, the chelator concentration will range from about0.1%-about 20% over a time course of about 5 minutes to about 24 hours,and in some embodiments, according to this aspect, the acidconcentration will range from about 0.01% to about 10%, over a timecourse of about 0.1 minute to about 24 hours.

According to this aspect, and in some embodiments, the contacting isconducted for a duration and under conditions, which vary as aconsequence of the desired final geometry of the scaffold.

In one embodiment of this invention, the term “portion” refers to alimited part of a whole. In one embodiment, the term “portion” withregard to the surface exposed as a consequence of the methods of thisinvention refers to a limited part of a whole exposed surface. Forexample, in one embodiment a portion of an exposed surface comprisesless than 100% of the exposed surface. In one embodiment a portion of anexposed surface comprises less than 90% of the exposed surface. In oneembodiment a portion of an exposed surface comprises less than 80% ofthe exposed surface. In one embodiment a portion of an exposed surfacecomprises less than 70% of the exposed surface. In one embodiment aportion of an exposed surface comprises less than 60% of the exposedsurface. In one embodiment a portion of an exposed surface comprisesless than 50% of the exposed surface. In one embodiment a portion of anexposed surface comprises less than 40% of the exposed surface. In oneembodiment a portion of an exposed surface comprises less than 30% ofthe exposed surface. In one embodiment a portion of an exposed surfacecomprises less than 20% of the exposed surface. In one embodiment aportion of an exposed surface comprises less than 10% of the exposedsurface. In one embodiment a portion of an exposed surface comprisesless than 1% of the exposed surface.

In one embodiment of this invention, the term “surface” refers to anexterior or upper boundary of an object.

In one embodiment of this invention, the term “exposed” refers to beingopen to the surrounding environment such that contact may occur betweena scaffold of this invention and the immersion media.

According to this aspect, and in other, embodiments, the solid formproduced by said process comprises at least two phases, which phasesdiffer in their pore volume, or which phases comprise voids which differin terms of the average diameter of said voids, or a combinationthereof. In some embodiments, the method as herein described is onemeans by which the scaffolds as described hereinabove may be prepared.

In some embodiments, this invention provides a scaffold producedaccording to a process of this invention.

In some embodiments, the methods of this invention result in scaffoldsproduced comprising phases, which differ in terms of the averagediameter of voids contained therein, or differ in terms of the porevolume within phases created in the scaffold thereby, or differ in termsof a combination thereof, which average diameter and/or pore volume aresmaller or larger than the ranges as described herein. It is to beunderstood that such scaffolds, as created by the methods of thisinvention represent envisioned embodiments of this invention and part ofthis invention.

In one embodiment, a scaffold of this invention comprises a solidthroughout a scaffold. One skilled in the art will recognize that solidscaffolding of this invention still comprises pore-like cavities and/orinterstices.

In one embodiment, a scaffold of this invention comprises a hollow alonga Cartesian coordinate axis of a scaffold. In one embodiment, the hollowis along a long axis of a scaffold of this invention. In one embodiment,the term “hollow” refers to a cavity within a scaffold of thisinvention. In one embodiment, the hollow comprises at least a singleopening in the scaffold such that the cavity is exposed to the externalenvironment. In one embodiment, the hollow provides additional exposedsurface area for a scaffold of this invention.

In some embodiments, the scaffolds of this invention will comprisemultiple hollows, which may be in any orientation, or in someembodiments, the scaffolds of this invention will comprise a network ofhollows within scaffolds, or in some embodiments, multiple scaffolds areimplanted into a repair site, wherein hollows of the scaffolds arealigned to form a network of hollows throughout the implanted scaffolds.

It will be appreciated by the skilled artisan that methods for selectivecreation of hollows or voids (which words may be used interchangeablythroughout) within the scaffolds of this invention may be prepared byany means known to the skilled artisan, for example, in accordance withthe methods as herein described, for example, by replacing immersiondipping of the portion of the scaffold with drip application of theimmersion solution to selectively create voids within the scaffolds ofthis application.

The exposed surface area of a scaffold of this invention provides alocation for mesenchymal stem cells, chondrocytes, osteoblasts, etc.,attachment, growth, proliferation or differentiation, or a combinationand a location for blood vessels formation. Therefore, the surface areaof a scaffold of this invention ultimately provides a beneficiallocation for regeneration of cartilage and/or bone tissue. In oneembodiment of this invention, a scaffold comprises a hollow, wherein thepresence of the hollow increases the exposed surface area of a scaffoldcompared to an analogous scaffold without a hollow.

In one embodiment of this invention, the scaffold comprises a polymercoating.

The term “polymer coating” refers, in some embodiments, to the presenceof a layer of polymeric material in association with at least a portionof the scaffolding material. In some embodiments, such coating may beover the entirety of the scaffold, and in some embodiments, such coatingmay penetrate to within the voids and/or pores and/or hollows of thescaffold. In some embodiments, such coating may be selectively appliedto a particular region of the scaffold, such that it creates a separatephase on the scaffold, and in some embodiments, such polymer may be soapplied that a thick polymer layer or phase is associated with a portionof a scaffold, thereby creating a separate polymer phase in associationwith the scaffolds as herein described. In some embodiments,biocompatible polymers are envisioned.

In one embodiment, the polymer coating strengthens the scaffold and insome embodiments, the polymer coating results in greater cellularattraction and attachment to the scaffolding, which in turn, inter alia,results in enhanced repair in terms of quantity, quality and timing ofrepair. In some embodiments, the polymer coating enhance cellsproliferation and/or differentiation into cartilage and/or bone which inturn, inter alia, results in enhanced repair in terms of quantity,quality and timing of repair.

In one embodiment of this invention, a polymer coating is permeable. Inone embodiment, the permeable polymer coating comprises a special porousmembrane. In one embodiment, the term “permeable” refers to having poresand openings. In one embodiment, the permeable polymer coating of thisinvention has pores and openings which allow entry of nutrients, atherapeutic compound, a cell population, a chelator, or a combinationthereof. In one embodiment, the permeable polymer coating of thisinvention has pores and openings which allow exit/release of nutrients,a therapeutic compound, a cell population, a chelator, or a combinationthereof.

In one embodiment, a polymer coating of this invention is discontinuous.In one embodiment, a region or a plurality of sub-regions of the coralof this invention comprise an absence of polymer coating, allowingdirect contact between the coral and the environment.

In some embodiments, the scaffold incorporates a biocompatible polymertherewithin, which is associated with the aragonite or calcitecomponent, via any physical or chemical association. In someembodiments, the polymer is a part of a hydrogel, which is incorporatedin the scaffolds of this invention. In some embodiments, suchhydrogel-containing scaffolds may thereafter be lyophilized ordessicated, and may thereafter be reconstituted.

In some embodiments of the scaffolds of this invention containing twoseparate phases, the biocompatible polymers are incorporated in thefirst phase alone or in the second phase alone.

Such polymer-containing scaffolds may be particularly suited forcartilage repair, regeneration or enhancement of formation thereof. Insome embodiments, according to this aspect, for example, in thetreatment of osteochondral defects, the coralline-based scaffolding isof a dimension suitable for incorporation within affected bone, andfurther comprises a polymer-containing phase, which phase, when insertedwithin the affected defect site, is proximal to affected cartilage. Inanother aspect and representing an embodiment of this invention, thescaffold comprises a polymer, which has permeated within the voids andpores of the scaffold, which scaffold is inserted within a site ofcartilage repair and which polymer facilitates cartilage growth,regeneration or healing of the defect site.

Such polymer-containing scaffolds may be particularly suited for bonerepair, regeneration or enhancement of formation thereof. In someembodiments, according to this aspect, for example, in the treatment ofbone breakage or fragmentation, disease or defect, the coralline-basedscaffolding is of a dimension suitable for incorporation within affectedbone, and further comprises a polymer, which polymer has permeatedwithin the voids and pores of the scaffold, which scaffold is insertedwithin the bone and which polymer facilitates bone growth, regenerationor healing of the defect site.

In one embodiment, a polymer coating of this invention comprises anatural polymer comprising, collagen, elastin, silk, hyaluronic acid,sodium hyaluronate, cross linked hyalronic acid, chitosan, cross linkedchitosan, alginate, calcium alginate, cross linked calcium alginate andany combinations thereof.

In one embodiment, the polymer comprises synthetically modified naturalpolymers, and may include cellulose, derivatives such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose estersand nitrocelluloses. Examples of suitable cellulose derivatives includemethyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxymethyl cellulose, cellulose triacetate andcellulose sulfate sodium salt.

In one embodiment, of this invention, a polymer comprises a syntheticbiodegradable polymer. In one embodiment of this invention, a syntheticbiodegradable polymer comprises alpha-hydroxy acids includingpoly-lactic acid, polyglycolic acid, enantioners thereof, co-polymersthereof, polyorthoesters, and combinations thereof.

In one embodiment, a polymer of this invention comprises apoly(cianoacrylate), poly(alkyl-cianoacrylate), poly(ketal),poly(caprolactone), poly(acetal), poly(α-hydroxy-ester),poly(α-hydroxy-ester), poly(hydroxyl-alkanoate),poly(propylene-fumarate), poly(imino-carbonate), poly(ester),poly(ethers), poly(carbonates), poly(amide), poly(siloxane),poly(silane), poly(sulfide), poly(imides), poly(urea),poly(amide-enamine), poly(organic acid), poly(electrolytes),polyp-dioxanone), poly(olefin), poloxamer, inorganic or organomatallicpolymers, elastomer, or any of their derivatives, or a copolymerobtained by a combination thereof.

In one embodiment, a polymer of this invention comprisespoly(D,L-lactide-co-glycolide) (PLGA). In another embodiment, thepolymer comprises poly(D,L-lactide) (PLA). In another embodiment, thepolymer comprises poly(D,L-glycolide) (PGA). In one embodiment, thepolymer comprises a glycosaminoglycan.

In one embodiment, the polymer comprises synthetic degradable polymers,which may include, but are not limited to polyhydroxy acids, such aspoly(lactide)s, poly(glycolide)s and copolymers thereof; poly(ethyleneterephthalate); poly(hydroxybutyric acid); poly(hydroxyvaleric acid);poly[lactide-co-(ε-caprolactone)]; poly[glycolide-co(ε-caprolactone)];poly(carbonate)s, poly(pseudo amino acids); poly(amino acids);poly(hydroxyalkanoate)s; poly(anhydrides); poly(ortho ester)s; andblends and copolymers thereof.

In one embodiment of this invention, a polymer comprises proteins suchas zein, modified zein, casein, gelatin, gluten, serum albumin,collagen, actin, α-fetoprotein, globulin, macroglobulin, cohesin,laminin, fibronectin, fibrinogen, osteocalcin, osteopontin,osteoprotegerin, or others, as will be appreciated by one skilled in theart. In another embodiment, a polymer may comprise cyclic sugars,cyclodextrins, synthetic derivatives of cyclodextrins, glycolipids,glycosaminoglycans, oligosaccharide, polysaccharides such as alginate,carrageenan (χ, λ, μ, κ), chitosane, celluloses, condroitin sulfate,curdlan, dextrans, elsinan, furcellran, galactomannan, gellan, glycogen,arabic gum, hemicellulose, inulin, karaya gum, levan, pectin, pollulan,pullulane, prophyran, scleroglucan, starch, tragacanth gum, welan,xanthan, xylan, xyloglucan, hyaluronic acid, chitin, or apoly(3-hydroxyalkanoate)s, such as poly(β-hydroxybutyrate),poly(3-hydroxyoctanoate) or poly(3-hydroxyfatty acids), or anycombination thereof.

In one embodiment, the polymer comprises a bioerodible polymer such aspoly(lactide-co-glycolide)s, poly(anhydride)s, and poly(orthoester)s,which have carboxylic groups exposed on the external surface as thesmooth surface of the polymer erodes, which may also be used. In oneembodiment, the polymer contains labile bonds, such as polyanhydridesand polyesters.

In one embodiment, a polymer may comprise chemical derivatives thereof(substitutions, additions, and elimination of chemical groups, forexample, alkyl, alkylene, hydroxylations, oxidations, and othermodifications routinely made by those skilled in the art), blends of,e.g. proteins or carbohydrates alone or in combination with syntheticpolymers.

In one embodiment of this invention, the polymer is biodegradable. Inone embodiment, the term “biodegradable” or grammatical forms thereof,refers to a material of this invention, which is degraded in thebiological environment of the subject in which it is found. In oneembodiment, the biodegradable material undergoes degradation, duringwhich, acidic products, or in another embodiment, basic products arereleased. In one embodiment, bio-degradation involves the degradation ofa material into its component subunits, via, for example, digestion, bya biochemical process. In one embodiment, biodegradation may involvecleavage of bonds (whether covalent or otherwise), for example in apolymer backbone of this invention. In another embodiment,biodegradation may involve cleavage of a bond (whether covalent orotherwise) internal to a side-chain or one that connects a side chainto, for example a polymer backbone.

In one embodiment, a coral of this invention is covalently associatedwith the polymer coating via the use of a cross-linking agent. In oneembodiment, the phrase “cross-linking agent” refers to an agent whichfacilitates the formation of a covalent bond between 2 atoms. In oneembodiment, the cross-linking agent is a zero-length cross-linkingagent.

In one embodiment, the cross-linking agent is (1 ethyl 3-(3dimethylaminopropyl) carbodiimide (EDAC), N-Sulfohydroxy succinamide (SulfoNHS), 5-iodopyrimidines, N-carbalkoxydihydroquinolines,pyrroloquinolinequinones, or a combination thereof.

In one embodiment, the cross-linking agent is a homobifunctionalcross-linker, such as, for example, a N-hydroxysuccinimide ester (e.g.disuccinimidyl suberate or dithiobis(succinimidylpropionate),homobifunctional imidoester (e.g. dimethyladipimidate or dimethylpimelimidate), sulfhydryl-reactive crosslinker (e.g.1,4-di-[3′-(2′-pyridyldithio)propionamido]butane), difluorobenzenederivative (e.g. 1,5-difluoro-2,4-dinitrobenzene), aldehyde (e.g.formaldehyde, glutaraldehyde), bis-epoxide (e.g. 1,4-butanedioldiglycidyl ether), hydrazide (e.g. adipic acid dihydrazide),bis-diazonium derivative (e.g. o-tolidine), bis-alkylhalide, or acombination thereof.

In one embodiment, the cross-linking agent is a heterobifunctionalcross-linker, such as, for example, an amine-reactive andsulfhydryl-reactive crosslinker (e.g. N-succinimidyl3-(2-pyridyldithio)propionate, a carbonyl-reactive andsulfhydryl-reactive crosslinker (e.g. 4-(4-N-maleimidophenyl)butyricacid hydrazide), or a combination thereof.

In some embodiments, the cross-linking agent is a trifunctionalcross-linkers, such as, for example,4-azido-2-nitrophenylbiocytin-4-nitrophenyl ester,sulfosuccinimidyl-2-[6-biotinamido]-2-(p-azidobenzamido)hexanoamido]ethyl-1,3′-dithiopropionate(sulfo-SBED), or a combination thereof.

In another embodiment, the cross-linking agent is an enzyme. In oneembodiment of this invention, the cross-linking agent comprises atransglutaminase, a peroxidase, a xanthine oxidase, a polymerase, or aligase, or a combination thereof.

The choice of concentration of the cross-linking agent utilized foractivity will vary, as a function of the volume, agent and polymerchosen, in a given application, as will be appreciated by one skilled inthe art.

In one embodiment, the association of a coral of this invention with apolymer coating of this invention comprises a physical and/or mechanicalassociation. For example, in one embodiment, a physical and/ormechanical association may comprise imbibing of any means, air drying,using a cross-linking agent, applying of heat, applying vacuum, applyinglyophilizing methods, freezing, applying mechanical forces or anycombination thereof, to promote the physical association between a coraland a polymer coating as described herein.

It will be apparent to one skilled the art that the physical and/orchemical properties of a polymer coating and components thereof mayinfluence methods of use of this invention and kits thereof, forinducing or enhancing cartilage and/or bone repair.

In one embodiment, the polymer coating of this invention has a thicknessof between 2.0 μm and 0.1 μm. In one embodiment, the polymer coating hasa thickness of about 1.0 μm. In one embodiment, the polymer coating ofthis invention has a thickness of between 10 μm and 50 μm. In oneembodiment, the polymer coating has a thickness of about 10-25, or about15-30, or about 25-50 μm.

In some embodiments, the polymer coating is a thin coating, which isassociated with the scaffolds of this invention and has a thickness asindicated hereinabove.

In some embodiments, the polymer coating is applied throughout thescaffolds of this invention, such that, in some embodiments, the poresand voids within the scaffolds of the invention may be filled with asherein described, and such polymer coatings may have a thickness ofabout 60-900 μm.

In some embodiments, the polymer coating is applied to a terminus or aportion of the coating forming an additional polymer phase on thescaffolds of the invention. According to this aspect, and in someembodiments, the polymer coating will have a thickness of between about0.1-10 mm.

In some embodiments, multiple scaffolds comprising polymer coatings areimplanted into a repair site, wherein the coating thickness of a firstscaffold may vary as compared to a coating thickness of a secondscaffold, implanted in the repair site. Variations in the coatingthickness may reflect the range described herein.

In one embodiment, the thickness of the polymer coating influencesphysical characteristics of a scaffold of this invention. For example,the thickness of a polymer coating may influence elasticity, tensilestrength, adhesiveness, or retentiveness, or any combination thereof ofa scaffold of this invention. In one embodiment, a polymer coatingincreases the elasticity of a scaffold of this invention. In oneembodiment, a polymer coating increases the tensile strength of ascaffold of this invention. In one embodiment, the adhesiveness of apolymer coating relates to adhesion of mesencynial stem cells, bloodvessels, tissue at a site of cartilage repair, cartilage tissue, or bonetissue, or a combination thereof. In one embodiment, a polymer coatingdecreases the adhesiveness of a scaffold of this invention. In oneembodiment, a polymer coating increases the adhesiveness of a scaffoldof this invention. One skilled in the art will recognize that a polymercoating may increase adhesiveness for an item while decreasingadhesiveness for another item. For example, in one embodiment, thepolymer coating increases adhesiveness for a mesenchymal stem cell anddecreases adhesiveness of an infective agent. In one embodiment, theretentiveness of a polymer coating relates to retention of a cellpopulation. In one embodiment, the cell population retained within apolymer coating is a mesenchymal stem cell population, chondrocytepopulation osteoblast population, etc. In one embodiment, theretentiveness of a polymer coating relates to retention of effectorcompounds.

In one embodiment, the thickness of the polymer coating influencesproliferation and/or differentiation of mesenchymal stem cells appliedto the scaffolds of this invention, or influences the activation ormigration of cells associated with cartilage and/or bone formation orrepair to the scaffolds of this invention, or a combination thereof.

In one embodiment of this invention, the cells as used in accordancewith the scaffolds, methods of use or kits of this invention, areengineered to express a desired product.

In one embodiment, a polymer coating of this invention comprises aneffector compound. In one embodiment, the effector compound is applieddirectly to a polymer coating of the scaffold of this invention. In oneembodiment, the effector compound comprises a component of a kit of thisinvention for use for incorporation into a scaffold of this invention asherein described. In one embodiment, the effector compound is applieddirectly to a polymer coating of this invention, without being dispersedin any solvent.

In one embodiment of this invention, the polymer coating comprises aneffector compound comprising a cytokine, a bone morphogenetic protein(BMP), growth factors, a chelator, a cell population, a therapeuticcompound, or an antibiotic, or any combination thereof.

In one embodiment, effector compounds for use in a scaffold and/or a kitof this invention and/or a method of this invention may comprise,inter-alia, a cytokine, a bone morphogenetic protein (BMP), growthfactor, a chelator, a cell population, a therapeutic compound, ananti-inflammatory compound, a pro-angiogenic compound or an antibiotic,or any combination thereof.

In one embodiment, the phrase “a cell population” refers to atransfected cell population, a transduced cell population, a transformedcell population, or a cell population isolated from a subject, or acombination thereof. In some embodiments, transfected, transduced ortransformed cells, may be incorporated into a polymer coat, or ascaffold of this invention, or a combination thereof.

In one embodiment, transfected, transduced or transformed cells, may beincorporated into a polymer coating, or a scaffold of this invention

In one embodiment, a cell population of this invention comprisesmesenchymal stem cells. In one embodiment, the mesenchymal stem cellsare transformed. In one embodiment, a cell population comprises cellsbeneficial in cartilage and/or bone formation and/or repair, such aschondroblasts or chondrocytes; fibrochondrocyte; osteocyte; osteoblast;osteoclast; synoviocyte; bone marrow cell; stromal cell; stem cell;embryonic stem cell; precursor cell, derived from adipose tissue;peripheral blood progenitor cell; stem cell isolated from adult tissue;genetically transformed cell; or a combination thereof. In anotherembodiment, a precursor cell may refer to a combination of chondrocytesand other cells; a combination of osteocytes and other cells; acombination of synoviocytes and other cells; a combination of bonemarrow cells and other cells; a combination of mesenchymal cells andother cells; a combination of stromal cells and other cells; acombination of stem cells and other cells; a combination of embryonicstem cells and other cells; a combination of precursor cells isolatedfrom adult tissue and other cells; a combination of peripheral bloodprogenitor cells and other cells; a combination of stem cells isolatedfrom adult tissue and other cells; and a combination of geneticallytransformed cells and other cells, the precursor cells for use in themethod of the present invention are prepared from an organ tissue of therecipient mammal (i.e. autologous), or a syngeneic mammal. In anotherembodiment, allogeneic and xenogeneic precursor cells may be utilized.

In one embodiment of this invention, the phrase “a therapeutic compound”refers to a peptide, a protein or a nucleic acid, or a combinationthereof. In another embodiment, the therapeutic compound is anantibacterial, antiviral, antifungal or antiparasitic compound. Inanother embodiment, the therapeutic compound has cytotoxic oranti-cancer activity. In another embodiment, the therapeutic compound isan enzyme, a receptor, a channel protein, a hormone, a cytokine or agrowth factor. In another embodiment, the therapeutic compound isimmunostimulatory. In another embodiment, the therapeutic compoundinhibits inflammatory or immune responses. In one embodiment, thetherapeutic compound comprises a pro-angiogenic factor.

In one embodiment, the phrase “a therapeutic compound”, refers to amolecule, which when provided to a subject in need, provides abeneficial effect. In some cases, the molecule is therapeutic in that itfunctions to replace an absence or diminished presence of such amolecule in a subject. In one embodiment, the molecule is a nucleic acidcoding for the expression of a protein is absent, such as in cases of anendogenous null mutant being compensated for by expression of theforeign protein. In other embodiments, the endogenous protein ismutated, and produces a non-functional protein, compensated for by theexpression of a heterologous functional protein. In other embodiments,expression of a heterologous protein is additive to low endogenouslevels, resulting in cumulative enhanced expression of a given protein.In other embodiments, the molecule stimulates a signaling cascade thatprovides for expression, or secretion, or others of a critical elementfor cellular or host functioning.

In another embodiment, the therapeutic compound may be natural ornon-natural insulins, amylases, proteases, lipases, kinases,phosphatases, glycosyl transferases, trypsinogen, chymotrypsinogen,carboxypeptidases, hormones, ribonucleases, deoxyribonucleases,triacylglycerol lipase, phospholipase A2, elastases, amylases, bloodclotting factors, UDP glucuronyl transferases, ornithinetranscarbamoylases, cytochrome p450 enzymes, adenosine deaminases, serumthymic factors, thymic humoral factors, thymopoietins, growth hormones,somatomedins, costimulatory factors, antibodies, colony stimulatingfactors, erythropoietin, epidermal growth factors, hepaticerythropoietic factors (hepatopoietin), liver-cell growth factors,interleukins, interferons, negative growth factors, fibroblast growthfactors, transforming growth factors of the α family, transforminggrowth factors of the β family, gastrins, secretins, cholecystokinins,somatostatins, serotonins, substance P, transcription factors orcombinations thereof.

In one embodiment, the effector compound comprises, an anti-helminth, anantihistamine, an immunomodulatory, an anticoagulant, a surfactant, anantibody, a beta-adrenergic receptor inhibitor, a calcium channelblocker, an ace inhibitor, a growth factor, a hormone, a DNA, an siRNA,or a vector or any combination thereof.

In one embodiment, the phrase “effector compound” refers to any agent orcompound, which has a specific purpose or application which is useful inthe treatment, prevention, inhibition, suppression, delay or reductionof incidence of infection, a disease, a disorder, or a condition, whenapplied to the scaffolds, kits and/or methods of this invention. Aneffector compound of this invention, in one embodiment, will produce adesired effect which is exclusive to the ability to image the compound.In some embodiments, the effector compound may be useful in imaging asite at which the compound is present, however, such ability issecondary to the purpose or choice of use of the compound.

In one embodiment of this invention, term “effector compound” is to beunderstood to include the terms “drug” and “agent”, as well, whenreferred to herein, and represents a molecule whose incorporation withinthe scaffold and/or kits of this invention, or whose use thereof, isdesired. In one embodiment, the agent is incorporated directly within ascaffold, and/or kit of this invention. In another embodiment; the agentis incorporated within a scaffold and/or kit of this invention, eitherby physical interaction with a polymer coating, a coral, or coralparticles of this invention, and/or a kit of this invention, orassociation thereto.

In one embodiment, compounds for use in a scaffold and/or a kit of thisinvention and/or a method of this invention may comprise, inter-alia, anantibody or antibody fragment, a peptide, an oligonucleotide, a ligandfor a biological target, an immunoconjugate, a chemomimetic functionalgroup, a glycolipid, a labelling agent, an enzyme, a metal ion chelate,an enzyme cofactor, a cytotoxic compound, a bactericidal compound, abacteriostatic compound, a fungicidal compound, a fungistatic compound,a chemotherapeutic, a growth factor, a hormone, a cytokine, a toxin, aprodrug, an antimetabolite, a microtubule inhibitor, a radioactivematerial, or a targeting moiety, or any combination thereof.

In one embodiment, the scaffolds and/or kits of this invention and/ormethods of this invention comprise or make use of an oligonucleotide, anucleic acid, or a vector. In some embodiments, the term“oligonucleotide” is interchangeable with the term “nucleic acid”, andmay refer to a molecule, which may include; but is not limited to,prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA,genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also refers to sequences that includeany of the known base analogs of DNA and RNA.

The scaffolds and/or kits of this invention and/or methods of use ofthis invention may comprise nucleic acids, in one embodiment, or inanother embodiment, the scaffolds and/or kits of this invention and/ormethods of use of this invention may include delivery of the same, as apart of a particular vector. In one embodiment, polynucleotide segmentsencoding sequences of interest can be ligated into commerciallyavailable expression vector systems suitable fortransducing/transforming mammalian cells and for directing theexpression of recombinant products within the transduced cells. It willbe appreciated that such commercially available vector systems caneasily be modified via commonly used recombinant techniques in order toreplace, duplicate or mutate existing promoter or enhancer sequencesand/or introduce any additional polynucleotide sequences such as forexample, sequences encoding additional selection markers or sequencesencoding reporter polypeptides.

In one embodiment, the scaffold of this invention incorporates stem orprogenitor or precursor cells. Such cells can be obtained directly froma mammalian donor, e.g., a patient's own cells, from a culture of cellsfrom a donor, or from established cell culture lines. In someembodiments, the mammal is a mouse, rat, rabbit, guinea pig, hamster,cow, pig, horse, goat, sheep, dog, cat, monkey, ape or a human. Cells ofthe same species and/or of the same immunological profile can beobtained by biopsy, either from the patient or a close relative. Usingstandard cell culture, techniques and conditions, the cells are thengrown in culture until confluent and used when needed. The cells may becultured until a sufficient number of cells have been obtained for aparticular application.

In one embodiment, the scaffold of this invention incorporates any cellwhich may participate in cartilage and/or bone formation or repair. Insome embodiments, such cells represent autografts, in that cells arecultured ex-vivo to seed the cells on the scaffolds of the invention,and such seeded scaffolds are implanted into the subject.

In some embodiments, such cells may represent allografts or xenografts,which may be incorporated within the scaffolds of this invention andimplanted within a site of repair.

In one embodiment, a coral of this invention comprises a cell populationfrom in vitro culture of the coral for a time period sufficient to seedthe cells in the coral. In one embodiment, the cell population is amesenchymal stem cell population, chondrocyte; fibrochondrocyte;osteocyte; osteoblast; osteoclast; synoviocyte; bone marrow cell;stromal cell; stem cell; embryonic stem cell; precursor cell, derivedfrom adipose tissue; peripheral blood progenitor cell; stem cellisolated from adult tissue; genetically transformed cell; or acombination thereof. In one embodiment, the mesenchymal stem cells;chondrocyte; fibrochondrocyte; osteocyte; osteoblast; osteoclast;synoviocyte; bone marrow cell; stromal cell; stem cell; embryonic stemcell; precursor cell, derived from adipose tissue; peripheral bloodprogenitor cell; stem cell isolated from adult tissue; geneticallytransformed cell; or a combination thereof seeded in vitro aretransformed. In one embodiment, the cell population comprises a cellpopulation beneficial for cartilage repair. In one embodiment, theculture comprises a chelator. In one embodiment of this invention, thechelator in a culture comprises a calcium chelator.

In one embodiment, a method of this invention induces or enhancescartilage and/or bone formation and/or repair, wherein the methodcomprises implanting in a subject, a scaffold of this invention within asite of cartilage and/or bone formation and/or repair, wherein a regionof the scaffold penetrates through a bone, resulting in the regioninserting within a bone marrow void, proximal to the site of cartilageand/or bone formation and/or repair.

In one embodiment, the phrase “cartilage repair” refers to restoring acartilage defect to a more healthful state. In one embodiment, restoringcartilage results in regeneration of cartilage tissue. In oneembodiment, restoring cartilage results in regeneration of a full orpartial thickness articular cartilage defect. In one embodiment,restoring cartilage results in complete or partial regeneration ofcartilage tissue at a site of cartilage repair. In one embodiment,cartilage repair may result in restoration/repair of missing ordefective bone tissue, wherein repair of a cartilage defect necessitatesremoval of bone tissue at a site of cartilage repair. In one embodiment,restoring cartilage results in regeneration of osteochondral defect. Inone embodiment, cartilage repair comprises restoring cartilage defectsof joints (e.g. knee, elbow, hip, shoulder joints), of ears, of a nose,or of a wind pipe.

In one embodiment, the phrase “bone repair” refers to restoring a bonedefect to a more healthful state. In one embodiment, restoring boneresults in regeneration of bone tissue. In one embodiment, restoringbone results in the filling in of any fracture or void within a bonetissue. In one embodiment, restoring bone results in complete or partialregeneration of bone tissue at a site of bone repair. In one embodiment,bone repair may result in restoration/repair of missing or defectivebone tissue. In one embodiment, bone repair comprises restoring bonedefects of any bone, as needed.

In some embodiments, the phrase “bone repair” refers to the treatment ofa subject with osteoporosis, Paget's disease, fibrous dysplasias, orosteodystrophies. In another embodiment, the subject has bone and/orcartilage infirmity. In another embodiment, the subject has other boneremodeling disorders include osteomalacia, rickets, rheumatoidarthritis, achondroplasia, osteochodrytis, hyperparathyroidism,osteogenesis imperfecta, congenital hypophosphatasia, fribromatouslesions, multiple myeloma, abnormal bone turnover, osteolytic bonedisease, periodontal disease, or a combination thereof. In oneembodiment, bone remodeling disorders include metabolic bone diseasewhich are characterized by disturbances in the organic matrix, bonemineralization, bone remodeling, endocrine, nutritional and otherfactors which regulate skeletal and mineral homeostasis, or acombination thereof. Such disorders may be hereditary or acquired and inone embodiment, are systemic and affect the entire skeletal system.

The scaffolds, kits and methods of the invention may also be used toenhance bone and/or cartilage formation in conditions where a boneand/or cartilage deficit is caused by factors other than bone remodelingdisorders. Such bone deficits include fractures, bone trauma, conditionsassociated with post-traumatic bone surgery, post-prosthetic jointsurgery, post plastic bone surgery, bone chemotherapy, post dentalsurgery and bone radiotherapy. Fractures include all types ofmicroscopic and macroscopic fractures. In one embodiment, some examplesof fractures includes avulsion fracture, comminuted fracture, transversefracture, oblique fracture, spiral fracture, segmental fracture,displaced fracture, impacted fracture, greenstick fracture, torusfracture, fatigue fracture, intraarticular fracture (epiphysealfracture), closed fracture (simple fracture), open fracture (compoundfracture) and occult fracture. In one embodiment, fractures meant to betreated using the methods of the present invention are non-unionfractures.

In one embodiment, the scaffolds, kits and methods of the invention mayalso be used to augment long bone fracture repair; generate bone insegmental defects; provide a bone graft substitute for fractures;facilitate tumor reconstruction or spine fusion; provide a localtreatment (by injection) for weak or osteoporotic bone, such as inosteoporosis of the hip, vertebrae, or wrist, or a combination thereof.In another embodiment, the scaffolds, kits and methods of the inventionmay also be used in a method to accelerate the repair of fractured longbones; treat of delayed union or non-unions of long bone fractures orpseudoarthrosis of spine fusions; induce new bone formation in avascularnecrosis of the hip or knee, or a combination thereof.

In one embodiment, a method of this invention comprises inducing andenhancing cartilage and/or bone repair wherein implanting a scaffold ofthis invention within a site of cartilage and/or bone repair influencesand improves cartilage and/or bone repair.

In one embodiment, a method of this invention induces or enhancescartilage and/or bone repair, wherein the scaffold attracts a populationof cells to the scaffold, thereby influencing or improving cartilageand/or bone repair.

The 3-D architecture and chemical composition of a scaffold of thisinvention are of great importance for specifically positioning andconfining a scaffold within a site of cartilage and/or bone repair; forcellular recognition, adhesion, proliferation and differentiation ofcell populations which induce or enhance cartilage and/or bone repair ora combination thereof.

In one embodiment, a scaffold of this invention utilized in a method ofthis invention comprises a seeded cell population prior to beingimplanted in a subject. In one embodiment, a method of this inventioninduces or enhances cartilage and/or bone repair, wherein implanting ina subject a scaffold of this invention promotes, adhesion, proliferationor differentiation, or a combination thereof of transformed mesenchymalstem cells. In one embodiment, a method of this invention induces orenhances cartilage and/or bone repair, wherein implanting in a subject ascaffold of this invention promotes blood vessel formation.

In one embodiment, a scaffold utilized in methods of this inventioncomprises at least a region which specifically positions and confinesthe coral scaffold at an optimal depth and angle within a site ofcartilage and/or bone repair, such that implanting the scaffold in asubject induces or enhances cartilage and/or bone repair. In oneembodiment, a scaffold utilized in methods of this invention comprisesat least a region which specifically positions and confines the coral atan optimal depth and angle within a site of cartilage and/or bonerepair, such that implanting the scaffold maximizes the contact areabetween a scaffold of this invention and a site of cartilage and/or bonerepair.

In one embodiment, a scaffold utilized in a method of the presentinvention may be used to adsorb or bind, and deliver, othertherapeutically active substances which assist in the cartilage and/orbone repair or regeneration process, or which have other desiredtherapeutic activity. Such substances include, by way of example, knownsynthetic or semisynthetic antibiotics which may be introduced into thepore cavities of the shaped product or structure, or a growth factorsuch as transforming growth factor or one of the bone morphogenicproteins which can be used to assist or promote bone growth.

In any of the embodiments herein, scaffolds for use in the methods ofthe present invention may further comprise, or be implanted with, othercompounds such as, for example, antioxidants, growth factors, cytokines,tibiotics, antiinflammatories, immunosuppressors, preservative, painmedication, other therapeutics, and excipient agents. In one embodiment,examples of growth factors that may be administered in addition to theHMG-CoA reductase inhibitor include, but are not limited to, epidermalgrowth factor (EGF), transforming growth factor-alpha (TGF-α),transforming growth factor-beta (TGF-β), human endothelial cell growthfactor (ECGF), granulocyte macrophage colony stimulating factor(GM-CSF), bone morphogenetic protein (BMP), nerve growth factor (NGF),vascular endothelial growth factor (VEGF), fibroblast growth factor(FGF), insulin-like growth factor (IGF), cartilage derived morphogeneticprotein (CDMP), platelet derived growth factor (PDGF), or anycombinations thereofxamples of antibiotics include antimicrobials andantibacterials.

In one embodiment, a method of this invention comprises implanting ascaffold of this invention in a subject afflicted with a cartilageand/or bone defect or disorder or disease.

In one embodiment, the term “implanting” refers to inserting and fixinga scaffold of this invention within a living site in a subject, the sitecomprising a site of cartilage and/or bone repair. In one embodiment, amethod of this invention implants a scaffold such a region of thescaffold now has access to mesenchymal stem cells, nutrients, bloodvessels, or effector compounds, or any combination of thereof. In oneembodiment, a method of this invention comprises implanting in a subjecta scaffold of this invention, wherein the method results in removing aregion of cartilage and/or bone and/or other tissue so that a region ofthe scaffold penetrates through the cartilage and/or bone and/or othertissue, and in some embodiments, reaches a bone marrow void.

A clinician skilled in the art will recognize that methods of thisinvention, which entail implanting a scaffold within a site of cartilageand/or bone repair, may require preparation of a site of cartilageand/or bone repair. These preparations may occur prior to implantationof a scaffold or simultaneously with implantation. For example,cartilage and/or bone tissue, and/or other tissues proximal to a site ofcartilage and/or bone repair may initially be drilled through to createa channel of dimensions appropriate for a scaffold used in the methodsof this invention. Then the scaffold is implanted within the site sothat a region of the scaffold penetrates the drilled cartilage and/orbone tissues. Alternatively, the scaffold may be attached to a tool ofthis invention capable of penetrating through cartilage and/or bone orother tissues, or a combination thereof. In this case, as the toolpenetrates through the cartilage and/or bone tissue, the attachedscaffold is simultaneously implanted.

In some embodiments, following implantation of the scaffold within arepair site, or several scaffolds within the repair site, the scaffoldis processed to optimize incorporation and optimal cartilage and/or bonerepair. In some embodiments, such processing may comprise cutting,sanding or otherwise smoothing the surface of the scaffold or scaffolds,for optimal repair.

In one embodiment, methods of this invention comprise implanting ascaffold in a human subject.

In one embodiment, methods of this invention may involve placement of ascaffold on a surface at site of cartilage and/or bone repair. In oneembodiment, methods of this invention may involve components of a tissuemilieu at a site of coral repair migrating to an exposed surface of acoral and contact between the coral of this invention would be made thuswith the environment.

In one embodiment, methods of this invention may involve implanting ascaffold so that raised exposed surfaces of the scaffold forcefullycontact the tissue at or adjacent to a site of cartilage and/or bonerepair. In this way, the exposed surface of coral now proximal to a siteof cartilage and/or bone repair is proximal to an environment comprisingcartilage tissue, bone tissue, bone marrow tissue, mesenchymal stemcells, nutrients, blood vessels or other effector compounds, or acombination thereof, which may be beneficial to cartilage and/or bonerepair.

In one embodiment of this invention, the phrases “long axis of thescaffold” and longitudinal axis of the scaffold” are usedinterchangeably and refer to a line extending parallel to the scaffoldlengthwise. The term “lengthwise” refers the direction of the length ofa scaffold. It may be that an original geometric shape has been cut toproduce a horizontal section of the original scaffold. In such instanceslengthwise should be viewed as being the original direction of lengthalong a scaffold.

It will be apparent to one skilled in the art that the physical and/orchemical properties of a scaffold of this invention and componentsthereof may influence methods of use of this invention and kits thereof,for inducing or enhancing cartilage and/or bone repair.

In one embodiment, methods of this invention for inducing or enhancingcartilage and/or bone repair utilize the 3-D geometry of a scaffold ofthis invention to provide for specifically positioning and confining thescaffold within a site of cartilage and/or bone repair.

In one embodiment, the term “proximal” refers to something beingsituated close to a particular locale. In one embodiment, a scaffold ofthis invention is forcibly held in position within a site of cartilageand/or bone repair by a raised region of the scaffold contacting tissuesituated at or proximal to a site of cartilage and/or bone repair.

One skilled in the art will recognize that the shape of a site ofcartilage and/or bone repair and the shape of a 3-D scaffold of thisinvention provide many different combinations for stably positioning ascaffold within a site of cartilage and/or bone repair. In oneembodiment, a scaffold of this invention is shaped prior to use inmethods of this invention for cartilage and/or bone repair. In oneembodiment, a scaffold of this invention is shaped concurrent to use inmethods of this invention for cartilage and/or bone repair. By shaping ascaffold concurrent with use of the scaffold in methods of thisinvention, the dimensions of the scaffold may be precisely selected forspecific positioning of the scaffold within a site of repair.

In one embodiment, methods of this invention comprise implanting ascaffold in a non-human mammalian and non-mammalian subject. In oneembodiment, methods of this invention comprise implanting a scaffold ina horse, a race horse, a cow, a steer, a pig, a rabbit, a goat, a sheep,a farm animal, a pet, a dog, a cat, a monkey, an ape, a bird and an aves

In one embodiment, methods of this invention are utilized for induced orenhanced repair of a cartilage and/or bone defect or disorder ordisease. In one embodiment, the cartilage defect results from a trauma,a tear, a sports injury, a full thickness articular cartilage defect, ajoint defect, or a repetitive stresses injury (e.g., osteochondralfracture, secondary damage due to cruciate ligament injury). In oneembodiment, the cartilage disorder comprises a disease of the cartilage.In one embodiment, methods of this invention induce or enhance cartilagerepair in osteoarthritis, rheumatoid arthritis, aseptic necrosis,osteochondritis dissecans, articular cartilage injuries, chondromalaciapatella, chondrosarcoma, chondrosarcoma—head and neck, costochondritis,enchondroma, hallux rigidus, hip labral tear, osteochondritis dissecans,torn meniscus, relapsing polychondritis, canine arthritis, fourthbranchial arch defect or cauliflower ear. In one embodiment, methods ofthis invention induce or enhance cartilage repair in degenerativecartilagenous disorders comprising disorders characterized, at least inpart, by degeneration or metabolic derangement of connective tissues ofthe body, including not only the joints or related structures, includingmuscles, bursae (synovial membrane), tendons, and fibrous tissue, butalso the growth plate, meniscal system, and intervertebral discs.

In one embodiment, a cartilage and/or bone defect or disorder or diseaserepaired by the methods of this invention utilizing a scaffold and/or atleast a tool of this invention, comprises a joint of a subject (e.g. aknee, elbow, ankle, shoulder, or hip joint), a rotator cup, an ear, anose, a windpipe, a pelvis, a spine, a rib, a jaw, a skull or any othersite of cartilage and/or bone defect or disorder or disease found withinthe subject.

In one embodiment, the 3-D shape and chemical composition of a scaffoldof this invention, used in the methods and/or kits of this inventionwill be determined by skilled clinicians, based on factors such as exactnature of the condition being treated, the severity of the condition,the age and general physical condition of the subject, body weight, andresponse of the individual subject, etc.

In one embodiment, the specific positioning of a scaffold of thisinvention during methods of this invention will be determined by skilledclinicians, based on factors such as exact nature of the condition beingtreated, the severity of the condition, the age and general physicalcondition of the subject, body weight, and response of the individualsubject, etc.

In one embodiment, methods of this invention are evaluated by examiningthe site of cartilage and/or bone tissue repair, wherein assessment isby histology, histochemistry, palpation, biopsy, endoscopy, arthroscopy,or imaging techniques comprising X-ray photographs, computerized X-raydensitometry, computerized fluorescence densitometry, CT, MRI or anothermethod known in the art, or any combination thereof.

In one embodiment, this invention provides an instrument to aid incartilage and/or bone repair comprising a tool to guide a scaffold ofthis invention to an optimal angle at a site of cartilage and/or bonerepair, a tool to guide a scaffold of this invention to an optimal angleat a site of cartilage and/or bone repair, a tool to deliver a scaffoldof this invention to a site of cartilage and/or bone repair, a tool toinsert a scaffold of this invention at a site of cartilage and/or bonerepair so that the scaffold penetrates through a cartilage and/or bone,and inserts within a bone marrow void, proximal to said site ofcartilage and/or bone repair, a tool to release a scaffold of thisinvention at a site of cartilage and/or bone repair, or a tool able toprovide a combination thereof, whereby the tool may be separated fromthe scaffold following placement of the scaffold within a site ofcartilage and/or bone repair.

In one embodiment, the instrument of this invention comprises at least asingle tool.

In one embodiment, methods of this invention utilize an instrument ofthis invention, wherein implanting a scaffold of this inventioncomprises specifically positioning and confining site the coral at anoptimal depth and angle within a site of cartilage and/or bone repair.

In some embodiments, such tools may comprise a tool for insertion of ascaffold into a repair site, which tool is specifically constructed tohold the scaffold and optimally position it within the site. In someembodiments, multiple tools, for different sized or shaped scaffolds maybe incorporated within kits of the invention, to accommodate theimplantation of varied scaffolds within a site or sites of cartilageand/or bone repair. In some embodiments, the kits of this invention willcomprise a tool to process the scaffold following insertion within thesite of repair, to affect a smooth optimal surface for optimal cartilageand/or bone repair. In some embodiments, the kits of this invention mayfurther comprise a tool for creating a void between the repair site anda source of mesenchymal stem cells. In some embodiments, the kits maycomprise a piece, which inserts within a common tool to effect such avoid, for example, a drill bit is included in the kits of this inventionof a size and depth to easily and appropriately drill through nearbybane in order that the scaffolding may be inserted in a site ofcartilage and/or bone repair, where at least a portion of the scaffold,or contiguous scaffolds insert within a site of cartilage and/or bonerepair and reach underlying bone marrow, to serve as a source formigrating mesenchymal stem cells to effect cartilage and/or bone repair.

One skilled in the art will recognize that the path created by drillingthrough tissue to reach a bone marrow void is such that it allows for ascaffold of this invention to reach the bone marrow void and be stablyimplanted at this site. The scaffold must be sufficiently secured withina site of cartilage and/or bone repair so that it does not get dislodgedas a joint articulates. A clinician skilled in the art will alsorecognize that the extent of a drilled path is such that a scaffold issecurely held but the path is not so extensive to incur increased damageto surrounding tissue.

Preparation of a site of cartilage and/or bone repair may also involveremoving damaged cartilage or bone tissue, or a combination thereof.Therefore, in one embodiment, a tool of this invention drills a pathsuch that damaged tissue at the site of repair or proximal to a site ofrepair is removed.

A tool of this invention may prepare the pathway a scaffold will follow,guide the scaffold being implanted, and implant the scaffoldconcurrently. By concurrently preparing the site and implanting thescaffold, the time of invasive or minimal-invasive surgery a subject issubjected to may be shortened.

In one embodiment, a region of the scaffold separates from the toolfollowing placement of the scaffold within the site of cartilage repair.In one embodiment, the region separates from the tool, whereinseparation of the tool from the scaffold comprises UV light-activatedseparation, LASER-activated separation, torsion-dependent separation orchemically-activated separation or a combination thereof. In oneembodiment, separation of the tool from the scaffold leaves behind thescaffold specifically positioned within a site of repair. The mechanismfor separation should also not cause additional trauma to a site ofrepair.

In one embodiment, separation of the tool from the scaffold results inthe scaffold being specifically positioned and confined at optimal depthand angle within a site of cartilage and/or bone repair. In oneembodiment, separation of the tool from the scaffold results in thescaffold being implanted in a subject within a site of cartilage and/orbone repair, wherein a region of the scaffold penetrates throughcartilage and/or bone, results in the region inserting within a bonemarrow void, proximal to the site of cartilage and/or bone repair.

In one embodiment, this invention provides a kit for repair of tissuecomprising the scaffold of this invention, at least a tool of thisinvention, and directions for utilizing the scaffold in tissue repair.

One skilled in the art will recognize that choice of a kit by a skilledclinician would be dependent upon factors such as exact nature of thecondition being treated, the severity of the condition, the age andgeneral physical condition of the subject, body weight, and response ofthe individual subject.

Thus, in one embodiment, the scaffold comprised in a kit of thisinvention comprises different sizes, shapes or chemical compositions, ora combination thereof. In one embodiment, this invention provides a kitfor cartilage and/or bone repair comprising a scaffold of thisinvention, at least a tool of this invention, and directions forutilizing the scaffold in cartilage repair.

Thus, the invention provides embodiments that include, inter alia,Embodiments 1-36 below:

Embodiment 1

-   -   A scaffold for tissue repair, said scaffold consisting        essentially of two phases wherein:        -   a first phase of said two phases comprises solid coral or            biolattice comprising hyaluronic acid; and        -   a second phase of said two phases comprises solid coral or            biolattice and said second phase further comprises a series            of hollows along a longitudinal axis in said second phase,            which is suited for enhanced vascularization or ease of            recruitment of progenitor cells, or a combination thereof;

Embodiment 2

-   -   The scaffold of embodiment 1, wherein said coral is from the        Porites species, Millepora species, or Acropora species;

Embodiment 3

-   -   The scaffold of embodiment 2, wherein said coral is Porites        lutea;

Embodiment 4

-   -   The scaffold of embodiment 2, wherein said coral is Millepora        dichotoma;

Embodiment 5

-   -   The scaffold of embodiment 2, wherein said coral is Acropora        grandis;

Embodiment 6

-   -   The scaffold of embodiment 1, wherein the first phase of said        scaffold further comprises a biocompatible polymer, comprising        the hyaluronic acid, and chitosan, cross linked chitosan,        alginate, calcium alginate, cross linked calcium alginate,        collagen and any combinations thereof;

Embodiment 7

-   -   The scaffold of embodiment 1, wherein said hyaluronic acid is        sodium hyaluronate or cross linked hyalronic acid;

Embodiment 8

-   -   The scaffold of embodiment 1, wherein the first phase of said        scaffold further comprises a biocompatible polymer, comprising        the hyaluronic acid, and an antiinflammatory compound, an        anti-infective compound, a growth factor, a chelator, an        antibiotic, a cell population, a pro-angiogenic factor or a        combination thereof;

Embodiment 9

-   -   The scaffold of embodiment 1, wherein said scaffold is seeded        with a cell population, which population comprises mesenchymal        stem cells, osteoblasts, osteocytes, osteoclasts, chondroblasts,        chondrocytes, fibroblasts, or a combination thereof;

Embodiment 10

-   -   The scaffold of embodiment 1, wherein said scaffold is        cylindrical in shape and has a diameter of about 5-15 mm, and a        height of about 5-25 mm;

Embodiment 11

-   -   The scaffold of embodiment 1, wherein said first phase is        inserted into a region which is proximal to cartilage and said        second phase is inserted into a region which is proximal to        subchondral bone;

Embodiment 12

-   -   A scaffold for the repair, regeneration or enhancement of        formation of cartilage, bone, or a combination thereof, which        scaffold consists of a solid form of aragonite or calcite and        further comprises:        -   at least a first phase, comprising voids having an average            diameter ranging from about 60-160 μm; and        -   at least a second phase, comprising voids having an average            diameter ranging from about 170-850 μm;

Embodiment 13

-   -   The scaffold of embodiment 12, further comprising a third phase,        comprising voids having an average diameter ranging from about        170-300 μm and said second phase comprises voids having an        average diameter ranging from about 350-850 μm and said third        phase is positioned between said first and second phase;

Embodiment 14

-   -   A scaffold for the repair, regeneration or enhancement of        formation of cartilage, bone, or a combination thereof, which        scaffold consists of a solid form of aragonite or calcite and        further comprises:        -   at least a first phase, comprising pores having a pore            volume ranging from about 35-55%; and        -   at least a second phase, comprising pores having a pore            volume ranging from about 56-95%;

Embodiment 15

-   -   The scaffold of embodiment 14, further comprising a third phase,        pores having a pore volume ranging from about 56-80%, wherein        said second phase comprises voids having an average pore volume        ranging from about 81-95% and said third phase is positioned        between said first and second phase;

Embodiment 16

-   -   The scaffold of embodiments 12-15, wherein said solid form is        isolated from a Pontes species, a Millepora species or an        Acropora species;

Embodiment 17

-   -   The scaffold of embodiment 16, wherein said solid form is        isolated from Pontes Lutea;

Embodiment 18

-   -   The scaffold of embodiment 16, wherein said solid form is        isolated from Acropora grandis;

Embodiment 19

-   -   The scaffold of embodiment 1, wherein said scaffold is of a        shape which accommodates a site of repair;

Embodiment 20

-   -   The scaffold of embodiments 12-15, wherein said scaffold        approximates the form of a cylinder, cone, screw, rectangular        bar, plate, disc, pyramid, granule, ball or cube;

Embodiment 21

-   -   The scaffold of embodiments 12-15, wherein said scaffold further        comprises a hollow or hollows along a Cartesian coordinate axis        of said scaffold in at least said first phase or at least said        second phase;

Embodiment 22

-   -   The scaffold of embodiments 12-15, wherein said scaffold further        comprises a biocompatible polymer;

Embodiment 23

-   -   The scaffold of embodiment 22, wherein said biocompatible        polymer is incorporated in said first phase alone or said second        phase alone;

Embodiment 24

-   -   The scaffold of embodiment 22, wherein said biocompatible        polymer comprises a natural polymer comprising a        glycosaminoglycan;

Embodiment 25

-   -   The scaffold of embodiment 22, wherein said glycosaminoglycan is        hyaluronic acid;

Embodiment 26

-   -   The scaffold of embodiments 12-15, wherein said scaffold further        comprises a cytokine, a bone morphogenetic protein (BMP), a        chelator, a therapeutic compound, or an antibiotic, or any        combination thereof;

Embodiment 27

-   -   The scaffold of embodiment 26, wherein said therapeutic compound        comprises an anti-inflammatory compound, an anti-infective        compound, a growth factor, a pro-angiogenic factor or a        combination thereof;

Embodiment 28

-   -   The scaffold of embodiments 12-15, wherein said scaffold is        seeded with a cell population, which population comprises        mesenchymal stem cells, osteoblasts, osteocytes, osteoclasts,        chondroblasts, chondrocytes, fibroblasts, or a combination        thereof;

Embodiment 29

-   -   The scaffold of embodiments 12-15, wherein said scaffold is        cylindrical in shape and has a diameter of about 5-15 mm, and a        height of about 5-25 mm;

Embodiment 30

-   -   A method of inducing or enhancing repair, regeneration or        enhancement of formation of cartilage, bone or a combination        thereof, said method comprising implanting in a subject, a        scaffold of embodiment 1 or embodiments 9-12 within a site in        need of repair, regeneration or enhancement of formation of        cartilage, bone or a combination thereof;

Embodiment 31

-   -   The method of embodiment 30, wherein said method comprises        exposing said site of cartilage repair, and optionally exposing        bone tissue located proximally to the site of cartilage repair        in said subject prior to implanting said scaffold;

Embodiment 32

-   -   The method of embodiment 31, further comprising the step of        affixing at least a portion of said scaffold within bone located        proximally to said site of cartilage repair;

Embodiment 33

-   -   The method of embodiment 32, wherein said method promotes        adhesion, proliferation or differentiation, or a combination        thereof, of a cell population to said scaffold;

Embodiment 34

-   -   The method of embodiment 32, further comprising seeding said        scaffold with mesenchymal stem cells prior to said implanting;

Embodiment 35

-   -   The method of embodiment 32, wherein said subject is afflicted        with a cartilage and/or bone defect or disorder or disease;

Embodiment 36

-   -   The method of embodiment 32, wherein said subject is a human        subject;

Embodiment 37

-   -   The method of embodiment 32, wherein said subject is an animal        subject;

Embodiment 38

-   -   The method of embodiment 32, wherein said cartilage defect or        disorder comprises a full or partial thickness articular        cartilage defect; osteochondral defect; osteoarthritis, a joint        defect or a defect resulting from trauma, sports, or repetitive        stress; and

Embodiment 39

-   -   The method of embodiment 32, wherein said scaffold is positioned        such that said at least a second porous phase is implanted        within or proximally to cartilage tissue and said at least a        first porous phase is implanted within or proximally to bone        tissue.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the scaffolds, kits, processand methods of the present invention without departing from the spiritor scope of the invention.

In some embodiments, the term “comprise” or grammatical forms thereof,refers to the inclusion of the indicated components of this invention,as well as inclusion of other active agents, and pharmaceuticallyacceptable carriers, excipients, emollients, stabilizers, etc., as areknown in the pharmaceutical industry.

In one embodiment, the present invention provides combined preparations.In one embodiment, the term “a combined preparation” defines especiallya “kit of parts” in the sense that the combination partners as definedabove can be used independently or in different combinations i.e.,simultaneously, concurrently, separately or sequentially.

EXAMPLES Example 1 Applications of Coralline-Based Scaffolding of thisInvention

Coralline-based scaffolding of this invention may be inserted intocartilage, bone or a combination thereof, in a subject in need thereof.

In some embodiments, such placement will include drilling in the area toexpose the site in which implantation is desired, and tight fitting ofthe scaffold within the defect/site.

For implantation for cartilage repair, regeneration, etc., scaffolds areimplanted in the desired cartilage site and within proximally locatedbone, so that, in this way, the coral scaffold is grafted through twotypes of tissue, cartilage and bone. FIG. 1 schematically depictsorientation of a cartoon of a scaffold of this invention within a siteof cartilage/bone repair.

Scaffolds may be prepared according to any embodiment as describedherein, as will be appreciated by the skilled artisan.

The scaffolds are envisioned for use in veterinary applications, as wellas in the treatment of human subjects. It is to be understood thatanimal studies may be undertaken to determine optimum configurations andimplantation parameters and procedures.

For example, animal studies may include implantation of a scaffold asdescribed herein within an animal subject and scaffolds are examined andobserved over an extended time period, post surgery. The untreated kneeof each animal is used as a control for comparisons following suchsurgeries. At appropriate intervals, animals are sacrificed andhistology performed. Appropriate time periods for examining the site ofcartilage repair are 2.5, 4, 9, 12, 26, 52 weeks post surgery. At thistime, the articular surfaces are photographed and tissue is removed fromthe site of repair and prepared for histological observations.Specifically, a block consisting of the grafted area and the surroundingtissue is removed using a fine saw. The material is further processedfor routine histology, which includes slow decalcification.

Example 2 Restoration of an Osteochondral Defect

Restoration of an osteochondral defect was performed in mature goatsusing rounded implants which were 6 mm in diameter and 8 mm in length. A5.5×8 mm core of cartilage and bone tissue was drilled out of, themedial femoral condyle of each goat (FIG. 2A) and the implant pressedfit into the site of cartilage and bone repair (FIGS. 2B and 2C).

Some animals harvested at 2.5 weeks post surgery exhibited signs thatthe implant was well incorporated into the native tissue and cartilagetissue was developed proximal to implant, moreover signs for:vascularisation can be seen (FIG. 2C).

A group of animals were sacrificed and tissue was harvested from theimplant site 9 weeks post surgery. H&E and Masson Trichrome histologicalevaluation of the tissue (FIGS. 3A and 3B, respectively) showed thatarea of the implant was replaced by newly formed cartilage and wovenbone and the cartilage was smooth and almost completely regenerated.Safranin O staining and probing for Collagen type II expression revealedthe existence of a homogeneous red band of cartilage covering normalbone (FIG. 4A), and collagen type II deposition along the band ofcartilage (FIG. 4B). The regenerated cartilage was virtuallyindistinguishable from the adjacent normal cartilage. The repair surfacewas smooth with no evidence of fibrillation. Moreover, there Wasevidence of complete closing of the defect at the level of the articularcartilage and evidence of transformation of mesenchymal cells tochondrocytes and osteoblasts with formation and remodeling (byosteoclasts) of new subchondral bone. FIG. 5A and FIG. 5B show an H & Estain of similar sections showing regenerated cartilage.

Example 3 Preparation of a Multiphasic Solid Aragonite Scaffold

To create a multi-phasic scaffold varying in terms of the pore volume(porosity) of each phase, and/or varying in terms of the diameter of thevoids present in each phase, plugs of 5.2 mm in diameter and 7.5 mm inlength were positioned within a silicon holder whereby only the top 1 mmof the plug was exposed, and the holder with the plug was placed in aninverted position, and immersed into the reaction mixture, such thatonly the top 1 mm of the plug was in direct contact with the mixture.

The plug was first immersed in a 5% disodium salt solution for two hoursat room temperature, followed by addition of a 99% formic acid solutionto yield a final concentration of 0.5%, where the plugs were immersedagain in the solution for an additional 20 minutes. The mixture wasdischarged and the plugs were washed in distilled water overnight, underconditions of approximately 0.2-0.00001 Bar pressure via the applicationof a vacuum following placement of the plug in a sealed container andapplying the vacuum to the chamber. Plugs were vacuum dried overnight atroom temperature.

FIG. 6A depicts an embodiment of a holder exposing a portion of a plugfor immersion as herein described.

Table 1 depicts the results of immersing 15 plugs isolated fromdifferent regions of the same piece of coral (Porites Lutea) processedas described in this example. A diamond saw was used to remove slicesfrom individual phases and the slices were processed for lightmicroscopy analysis and an optical bifocal microscope was used to imagethe scaffold; and images were captured and void diameters were assessedfor size by standard methodology.

30 voids were identified in three different implants, within the regionof the plug immersed within the reaction mixture, 41 voids wereidentified in four different implants within the region locatedproximally to the immersion region and 46 voids were identified in fourdifferent implants within the region located distally to the immersionregion and the diameter of each void was determined. The result of thesedeterminations is presented in Table 1 hereinbelow:

TABLE 1 Immersed region of the implant Proximal region Distal regionMedian 700 200 110 Std. Dev. 50.2 69.8 39.9

FIGS. 6B and 6C depict light microscopy images of the top portion (panelB) cut from the plug (panel C) and visualized at higher magnificationwhere the size of the voids can be ascertained.

In terms of pore volume (porosity), the immersed portion exhibited fromabout 85-90% of the plug being porous, the proximal region thereto(having a length of about 0.5-1 mm along a longitudinal axis, locatedproximally to the immersed region) exhibiting about 65-75% porosity,while the distal region exhibited about 45-50% porosity.

Porosity was derived, as described (Karageorgiou V, Kaplan D. (2005)“Porosity of 3D biomaterial scaffolds and osteogenesis” Biomaterials.;26(27):5474-91)

The porosity level can be controlled by parameters such as the type andconcentration of the chelator utilized, the type and concentration ofthe acid utilized, temperature at which the method is conducted and timeof reaction, while the size of the entire enlarged porous phase can becontrolled by the length of the plug which is placed in direct contactwith the reaction mixture.

Controlling of the porosity and the size of the different phases willallow for the design of implants for different purposes exhibitingdifferent strengths, physical and structural characteristics which havethe potential to mimic different native bones and cartilage structures.This control will provide a higher fitness of the implant to the exactdesired location of implantation as is required from each specificcartilage/bone or only bone defect.

Example 4 Scaffolds of Aragonite Impregnated with Hyaluronic Acid areMore Chondrogenic than Aragonite-Based Scaffolds

In order to evaluate the chondrogenic potential of the chondral phase ofan implant, aragonite-based scaffolds were compared to aragoniteimpregnated with hyaluronic acid scaffolds. In vitro assays wereconducted using the murine mesenchymal stem cells (MSCs) ATCC/CRL-12424and their differentiation toward a chordrogenic lineage was assessed.5000 MSCs were seeded onto 1 mg of small particles (˜1 mm in size) ofcoralline-based (sp. Porites Lutea) aragonite with or without hyaluronicacid (NaHA 1%). A third group without any implants served as control.The methods of the scaffold preparation were described herein. Thehyaluronic acid (HA) used was an injectable gel of 1% sodium hyaluronatemarketed as Arthrease, manufactured by Bio-Technology General (Israel)LTD.

Each particle was individually cultured and seeded separately. Cellswere grown in supplemented DMEM medium, without the addition of anyinductive chondrogenic agents. The medium was replaced every 2-3 daysfor a period of 21 days. Care was taken during media replacement to notdisturb the particles in the cultures. The assay was performed in threetriplicates.

Following one, two and three weeks in culture, the MSCs differentiationwas assayed by staining of the culture with Safranin O/Fast Greenstaining [Kahveci Z, Minbay F Z, Cavusoglu L (2000) Safranin O stainingusing a microwave oven. Biotech Histochem. 75(6):264-8] of cells fixedwith 4% glutaraldegyde solution. Digital images of the stained cellswere processed.

FIG. 7 shows Fast Green staining of cytoplasm of all the cells in abright green color while Safranin O staining of glycosaminiglycans (GAG)secreted by chondrocytes into the extracellular matrix of the cells isevident by a characteristic pink color.

The images were analyzed using Image) software. The color intensity wasused to estimate chondrogenesis. Each image was analyzed for the area ofstained cells with Safranin O (8A) and its integrated density (8B) ofpink color—it was calculated by counting the number of the pink coloredpixels in a specified area of the image, excluding the area of theparticles from the calculation.

During the entire study aragonite impregnated with hyaluronic acidshowed greater chondrogenesis in comparison to aragonite alone both interms of the stained area (amount of cells) and the color intensity(amount of GAG at the ECM).

Control assay included the assay of cells cultured identically, in theabsence of any scaffold, which showed significantly less characteristictoward the GAG staining.

The morphology of the cells was visualized using field emission ScaningElectron Microscopy (JEOL, JSM-7400F). MSCs seeded on the aragonite withhyaluronic acid exhibited a round morphology and developed denseextracellular matrix, which is typical for matured chondrocytes (FIGS.9C-D). In contrast, the MSCs, grown on aragonite without HA, showedflattened, fibroblast-like morphology (FIGS. 9A-B).

The aragonite-HA complex thus enabled MSC adherence, proliferation anddifferentiation toward a chondrogenic phenotype.

The chondrogenic potential of the aragonite impregnated with hyaluronicacid was demonstrated herein to provide superior chondrogenesis, therebysupporting the scope of the invention directed to abi-phasic implantwhere the chondral phase is composed of aragonite with holes/voids thatare impregnated with a biocompatible polymer such as hyaluronic acid,and a bone phase which is composed of aragonite or calcite alone.

Example 5 Aragonite and Calcite-Based Scaffolds are Chondrogenic In Vivo

Implantation may be at any suitable location, for example, for kneejoint repair, implantation may be within the Medial Femoral Condyle(MFC), Lateral Femoral Condyle (LFC), Patela, Trochlear Groove (TG) andthe Tibia.

Model systems using sheep, goats or horses may be utilized to testcertain embodied scaffolds of this invention. In the chosen implantationlocation, for example in the load bearing area of the MFC a defect ismade using a punch. The dimensions of the defect are measured, forexample, 5-10 mm and 6-12 mm in diameter and depth respectively.

The diameter of the implant will be appropriate for the diameter of theosteochondral defect being tested. For example, a chosen diameter of animplant may be 6 mm, to covet 5.8 mm diameter of the defect in order toassure good fixation to the defect in a press fit manner. A secondlocation in a non weight bearing place may be chosen, for comparison,for example within the TG.

In order to treat large cartilage lesions several implants, having thesame or different geometrical shapes and properties, can be introducedin order to fill the defect. The implantation can be performedarthroscopically or by an open incision (arthrotomy).

X-ray, CT or MRI imaging may be performed to verify the position of theimplants.

Example 6 Preparation of a Bi-Phasic Aragonite Scaffold ComprisingHyaluronic Acid at the Chondral Phase and Vascularization Channels atthe Bone Phase, for Packaging and Distribution

Preparation of the aragonite core scaffold: Coral from the hydrocoralPorites lutea which has an average pore size of 100-150 μm is harvestedevaluated visually for its appearance, density, porosity and issubjected to FTIR analysis. Amino acid quantification may also bedetermined. Coral is then immersed in 5% sodium hypochlorite for removalof external organic tissue.

Without being bound by theory, one means by which superior scaffoldingis produced by this process is a result of the penetration deep withinthe coral, whereas immersion processes or application of positivepressure during purification allows for air bubbles to remain trappedwithin the pore network of the scaffold, resulting in poor accessibilityof the solvents to inner compartments of the coral. Moreover, thepurification process facilitates removal of the oxidizing agentsemployed in a most thorough manner, a clearly desirable result forscaffolding later implanted in living beings.

A saw, for example, a diamond disk saw, is used to remove the outercoral layer, and plugs of the desired dimensions are cut from a largercoral block. A series of holes are then drilled through part of theplugs, comprising the second phase, which phase is intended to beimplanted within a site in need of bone repair or regeneration, oranchoring within bone, for eventual replacement with bone tissue,obtained to a desired depth and in a desired pattern/number, etc See forexample, FIG. 10.

Organic matter is removed from the coral as follows: the plugs are firstexposed to fluid containing an oxidizing agent under negative pressure,for example, a 5% sodium hypochlorite solution for 30 minutes, 3exchanges at temperature range RT at 50° C., and subatmospheric pressureusing vacuum pressure ranging from 0.2-0.00601 Bar. The plugs are thenexposed to a 10% solution of hydrogen Peroxide for 15 minutes attemperature range RT at 50° C., and subatmospheric pressure using vacuumpressure ranging from 0.2-0.00001 Bar. The cleaned plugs are then washedin distilled water for 30 minutes, 3 exchanges at pressure temperaturerange RT at 50° C., and subatmospheric pressure using vacuum pressureranging from 0.2-0.00001 Bar.

The coral is sterilized by exposure to gamma radiation at a strength ofat least 22.5 kGy and can then be stored aseptically, in packagingmaterial.

Sodium hyalronate 1% (hyaluronic acid 1% solution in phosphate bufferedsaline, described hereinabove) is applied apically to the plug. Anapical portion of the plug is constrained within a ring, for example asilicon ring, which spans a region above the terminus of the plugcreating a reservoir at the terminus of the plug. Hyaluronic acidsolution is then applied to the first phase, i.e. the phase which isinserted/implanted within cartilage, for example, 70 ml of the solutionis applied over a 6 mm in diameter plug to produce 2 mm homogenous phaseof coral impregnating the hyaluronic acid to treat the chondral defect,which is immobilized in a silicon ring which spans the plug terminus and4 mm above the terminus. The application of the hyaluronic acid solutionproceeds for 45-60 minutes, and the ring is then removed, the plug isinserted in a sterile packaging and sealed. The sealed packaging is thensubjected to evaporation under vacuum conditions to dry out the NaHA 1%solution to form dried HA coating of the chondral phase of the plug(e.g. by lyophilization/dessication). A sterile pack containing thedried product is thereby obtained.

FIG. 11 schematically depicts an embodied scaffold of the invention,wherein a first phase 11-10 comprising hyaluronic acid is prepared asherein described. This first phase is shown atop a second phase 11-20comprised of aragonite alone, which phase has been physically modifiedto comprise a series of vacularization channels 11-30. The terminus ofthe second phase may be tapered 11-40 to allow for easy tight fitting ofthe scaffold in question.

It will be understood by those skilled in the art that various changesin form and details may be made therein without departing from thespirit and scope of the invention as set forth in the appended claims.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed in the scope of the claims.

In one embodiment of this invention, “about” refers to a quality whereinthe means to satisfy a specific need is met, e.g., the size may belargely but not wholly that which is specified but it meets the specificneed of cartilage repair at a site of cartilage repair. In oneembodiment, “about” refers to being closely or approximate to, but notexactly. A small margin of error is present. This margin of error wouldnot exceed plus or minus the same integer value. For instance, about 0.1micrometers would mean no lower than 0 but no higher than 0.2. In someembodiments, the term “about” with regard to a reference valueencompasses a deviation from the amount by no more than 5%, no more than10% or no more than 20% either above or below the indicated value.

In the claims articles such as “a,”, “an” and “the” mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” or “and/or” betweenmembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention provides, in various embodiments, all variations,combinations, and permutations in which one or more limitations,elements, clauses, descriptive terms, etc., from one or more of thelisted claims is introduced into another claim dependent on the samebase claim unless otherwise indicated or unless it would be evident toone of ordinary skill in the art that a contradiction or inconsistencywould arise. Where elements are presented as lists, e.g. in Markushgroup format or the like, it is to be understood that each subgroup ofthe elements is also disclosed, and any element(s) can be removed fromthe group. It should be understood that, in general, where theinvention, or aspects of the invention, is/are referred to as comprisingparticular elements, features, etc., certain embodiments of theinvention or aspects of the invention consist, or consist essentiallyof, such elements, features, etc. For purposes of simplicity thoseembodiments have not in every case been specifically set forth in haecverba herein. Certain claims are presented in dependent form for thesake of convenience, but Applicant reserves the right to rewrite anydependent claim in independent format to include the elements orlimitations of the independent claim and any other claim(s) on whichsuch claim depends, and such rewritten claim is to be consideredequivalent in all respects to the dependent claim in whatever form it isin (either amended or unamended) prior to being rewritten in independentformat.

What is claimed is:
 1. A scaffold for tissue repair, said scaffoldconsisting essentially of two phases wherein: a first phase of said twophases comprises solid coral comprising a biocompatible polymer; and asecond phase of said two phases comprises solid coral and said secondphase further comprises a series of hollows along a longitudinal axis insaid second phase, wherein said first phase does not comprise a seriesof hollows along a longitudinal axis said second phase does not comprisea biocompatible polymer and said scaffold is cut from or shaped from asingle piece of coral, and wherein said biocompatible polymer in saidfirst phase comprises a glycosaminoglycan comprising hyaluronic acid,sodium hyaluronate, cross-linked hyaluronic acid, or a combinationthereof.
 2. The scaffold of claim 1, wherein the biocompatible polymerin the first phase further comprises another glycosaminoglycan,chitosan, cross linked chitosan, alginate, calcium alginate, crosslinked calcium alginate, collagen, elastin, silk or a combinationthereof.
 3. The scaffold of claim 2, wherein said glycosaminoglycan ishyaluronic acid.
 4. The scaffold of claim 1, wherein the said scaffoldfurther comprises an anti-inflammatory compound, an anti-infectivecompound, a growth factor, a chelator, an antibiotic, a cell population,a pro-angiogenic factor or a combination thereof.
 5. The scaffold ofclaim 4, wherein said growth factor is wherein said growth factor isepidermal growth factor (EGF), transforming growth factor-beta (TGF-β),human endothelial cell growth factor (ECGF), granulocyte macrophagecolony stimulating factor (GM-CSF), bone morphogenetic protein (BMP),nerve growth factor (NGF), vascular endothelial growth factor (VEGF),fibroblast growth factor (FGF), insulin-like growth factor (IGF),cartilage derived morphogenetic protein (CDMP), platelet derived growthfactor (PDGF), or a combination thereof.
 6. The scaffold of claim 1,wherein said scaffold is seeded with a cell population, which populationcomprises mesenchymal stem cells, osteoblasts, osteocytes, osteoclasts,chondroblasts, chondrocytes, fibroblasts, or a combination thereof. 7.The scaffold of claim 1, wherein said biocompatible polymer isincorporated within at least a portion of the first phase of saidscaffold.
 8. The scaffold of claim 1, wherein said scaffold approximatesthe form of a cylinder, cone, tac, screw, rectangular bar, plate, disc,pyramid, granule, ball or cube.
 9. The scaffold of claim 1, wherein thescaffold assumes a shape to approximate a meniscus for a knee or elbow;a joint; an articular surface of a bone, a portion of a rib cage, a hip,a pelvis, an ear, a nose, a ligament, a bronchial tube or anintervertebral disc.
 10. The scaffold of claim 1, wherein said scaffoldis cylindrical in shape and has a diameter of about 5-40 mm, and aheight of about 5-25 mm.
 11. The scaffold of claim 1, wherein saidbiocompatible polymer is in the form of a polymer coating, forming apolymer layer associated with a portion of said first phase of saidscaffold.
 12. The scaffold of claim 11, wherein said polymer coating ispresent at a terminus of said scaffold and said coating has a thicknessof between about 0.1-10 mm.
 13. A method of inducing or enhancingrepair, regeneration or enhancement of formation of cartilage, bone or acombination thereof, said method comprising implanting in a subject, ascaffold of claim 1 within a site in need of repair, regeneration orenhancement of formation of cartilage, bone or a combination thereof insaid subject.
 14. The method of claim 13, wherein said cartilage defector disorder comprises a full or partial thickness articular cartilagedefect; osteochondral defect; osteochondritis dissecans; osteoarthritis,a joint defect or a defect resulting from trauma, sports, or repetitivestress.
 15. The method of claim 13, wherein said scaffold is positionedsuch that said first phase is implanted within or proximally tocartilage tissue and said second phase is implanted within or proximallyto bone tissue.
 16. A kit for repair of cartilage and bone comprisingthe scaffold of claim 1; directions for utilizing said scaffold intissue repair; and optionally a tool for optimal insertion of saidscaffold, optionally wherein said kit comprises a series of scaffolds ofdifferent sizes, shapes or a combination thereof.
 17. The scaffold ofclaim 3, wherein said glycosaminoglycan both is hyaluronic acid andsodium hyaluronate.
 18. The scaffold of claim 3, wherein saidglycosaminoglycan is both hyaluronic acid and cross linked hyalronicacid.